Method for detecting methylation of colorectal cancer specific methylation marker gene for colorectal cancer diagnosis

ABSTRACT

The present disclosure relates to a method for detecting methylation of the bowel-cancer-specific methylation marker GPM6A (NM_201591, glycoprotein M6A) gene in order to diagnose bowel cancer, and more specifically relates to a method for providing information for diagnosing bowel cancer by detecting the methylation of a bowel-cancer-specific marker gene that is specifically methylated in bowel cancer cells. The method for detecting methylation and a diagnostic composition, kit and nucleic-acid chip according to the present disclosure can be used to advantage in diagnosing bowel cancer more accurately and quickly than by normal methods as they permit bowel cancer to be diagnosed at the initial genetic transformation step and so allow early diagnosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part application under 35 U.S.C. §120 of U.S. patent application Ser. No. 15/661,054 filed Jul. 27, 2017, which in turn is a continuation-in-part application of U.S. patent application Ser. No. 15/010,379 filed Jan. 29, 2016, now U.S. Pat. No. 9,752,197, which in turn is a continuation-in-part application of U.S. patent application Ser. No. 13/994,732 filed Jun. 15, 2013, now U.S. Pat. No. 9,315,870, which in turn is a U.S. national stage under the provisions of 35 U.S.C. §371 of International Patent Application No. PCT/KR2011/009710 filed Dec. 16, 2011, which in turn claims priority of Korean Patent Application No. 10-2010-129208 filed Dec. 16, 2010. The disclosures of such U.S. patent applications, international patent application, and Korean priority patent application are hereby incorporated herein by reference in their respective entireties, for all purposes.

TECHNICAL FIELD

The present disclosure relates to a method for detecting the methylation of colorectal cancer-specific marker genes for colorectal cancer diagnosis, and more particularly to a method of detecting the methylation of a colorectal cancer-specific marker gene, which are methylated specifically in colorectal cancer cells, to provide information for diagnosing colorectal cancer.

BACKGROUND ART

In current clinical practice, the diagnosis of cancer is confirmed by performing tissue biopsy after history taking, physical examination and clinical assessment, followed by radiographic testing and endoscopy if cancer is suspected. However, the diagnosis of cancer by the existing clinical practices is possible only when the number of cancer cells is more than a billion and the diameter of cancer is more than 1 cm. In this case, the cancer cells already have metastatic ability, and at least half thereof have already metastasized. Meanwhile, tumor markers for monitoring substances that are directly or indirectly produced from can92cers are used in cancer screening, but they cause confusion due to limitations in accuracy, since up to about half thereof appear normal even in the presence of cancer, and they often appear positive even in the absence of cancer. Furthermore, the anticancer agents that are mainly used in cancer therapy have the problem that they show an effect only when the volume of cancer is small.

Recently, genetic analysis has been actively attempted to diagnose cancer. The simplest typical method is to detect the presence of ABL:BCR fusion genes (the genetic characteristic of leukemia) in blood by PCR. The method has an accuracy rate of more than 95%, and after the diagnosis and therapy of chronic myelocytic leukemia using this simple and easy genetic analysis, this method is being used for the assessment of the result and follow-up study. However, this method has a shortcoming in that it can be applied only to some blood cancers.

Furthermore, another method has been attempted, in which the presence of genes expressed by cancer cells is detected by RT-PCR and blotting, thereby diagnosing cancer cells present in blood cells. However, this method has shortcomings in that it can be applied only to some cancers, including prostate cancer and melanoma, has a high false positive rate. In addition, it is difficult to standardize detection and reading in this method, and its utility is also limited (Kopreski, M. S. et al., Clin. Cancer Res., 5:1961, 1999; Miyashiro, I. et al., Clin. Chem., 47:505, 2001).

Recently, genetic testing that uses a DNA in serum or blood plasma has been actively attempted. This is a method of detecting a cancer-related gene that is isolated from cancer cells and released into blood and present in the form of a free DNA in serum. It is found that the concentration of DNA in serum is increased by a factor of 5-10 times in actual cancer patients as compared to that of normal persons, and such increased DNA is released mostly from cancer cells. The analysis of cancer-specific gene abnormalities, such as the mutation, deletion and functional loss of oncogenes and tumor-suppressor genes, using such DNAs isolated from cancer cells, allows the diagnosis of cancer. In this effort, there has been an active attempt to diagnose lung cancer, head and neck cancer, breast cancer, colorectal cancer, and liver cancer by examining the promoter methylation of mutated K-Ras oncogenes, p53 tumor-suppressor genes and p16 genes in serum, and the labeling and instability of microsatellite (Chen, X. Q. et al., Clin. Cancer Res., 5:2297, 1999; Esteller, M. et al., Cancer Res., 59:67, 1999; Sanchez-Cespedes, M. et al., Cancer Res., 60:892, 2000; Sozzi, G. et al., Clin. Cancer Res., 5:2689, 1999).

Meanwhile, in samples other than blood, the DNA of cancer cells can also be detected. A method has been attempted in which the presence of cancer cells or oncogenes in sputum or bronchoalveolar lavage of lung cancer patients is detected by a gene or antibody test (Palmisano, W. A. et al., Cancer Res., 60:5954, 2000; Sueoka, E. et al., Cancer Res., 59:1404, 1999). Additionally, other methods of detecting the presence of oncogenes in feces of colorectal cancer patients (Ahlquist, D. A. et al., Gastroenterol., 119:1219-27, 2000) and detecting promoter methylation abnormalities in urine and prostate fluid (Goessl, C. et al., Cancer Res., 60:5941, 2000) have been attempted. However, in order to accurately diagnose cancers that cause a large number of gene abnormalities and show various mutations characteristic of each cancer, a method in which a large number of genes are simultaneously analyzed in an accurate and automatic manner is required. However, such a method has not yet been established.

Accordingly, methods of diagnosing cancer by measuring DNA methylation have recently been proposed. When the promoter CpG island of a certain gene is hyper-methylated, the expression of such a gene is silenced. This is interpreted to be a main mechanism by which the function of this gene is lost even when there is no mutation in the protein-coding sequence of the gene in a living body. In addition, this is analyzed as a factor by which the function of a number of tumor-suppressor genes in human cancer is lost. Thus, analysis of the methylation of the promoter CpG island of tumor-suppressor genes is very helpful in cancer research. An active attempt has been made to analyze the methylation of the promoter CpG island by methods such as methylation-specific PCR (hereinafter, referred to as “MSP”) or automatic base sequencing and to use the analysis results for the diagnosis and screening of cancer.

A significant number of diseases are caused by genetic abnormalities, and the most frequent form of genetic abnormality is a change in the coding sequence of a gene. This genetic change is referred to as mutation. When any gene has a mutation, the structure and function of a protein encoded by the gene change, resulting in abnormalities and deletions, and this mutant protein causes disease. However, an abnormality in the expression of a specific gene can cause disease even in the absence of a mutation in the gene. A typical example thereof is methylation in which a methyl group is attached to the transcription regulatory region of a gene, that is, the cytosine base of the promoter CpG islands, and in this case, the expression of the gene is silenced. This is known as epigenetic change. This is transmitted to offspring and results in the loss of the expression of the relevant protein in the same manner as mutation. Most typically, the expression of tumor suppressor genes is silenced by the methylation of promoter CpG islands in cancer cells, resulting in carcinogenesis (Robertson, K. D. et al., Carcinogensis, 21:461, 2000).

During a cancer-causing process, methylation is found in promoter CpG islands, and the restriction on the corresponding gene expression occurs. Particularly, if methylation occurs in the promoter CpG islands of tumor-suppressor genes that regulate cell cycle or apoptosis, restore DNA, are involved in the adhesion of cells and the interaction between cells, and/or suppress cell invasion and metastasis, such methylation blocks the expression and function of such genes in the same manner as the mutations of a coding sequence, thereby promoting the development and progression of cancer. In addition, partial methylation also occurs in the CpG islands according to aging.

An interesting fact is that, in the case of genes whose mutations are attributed to the development of cancer in congenital cancer but do not occur in acquired cancer, the methylation of promoter CpG islands occurs instead of mutation. Typical examples include the promoter methylation of genes, such as acquired renal cancer VHL (von Hippel Lindau), breast cancer BRCA1, colorectal cancer MLH1, and stomach cancer E-CAD. In addition, in about half of all cancers, the promoter methylation of p16 or the mutation of Rb occurs, and the remaining cancers show the mutation of p53 or the promoter methylation of p73, p 14 and the like.

An important fact is that an epigenetic change caused by promoter methylation causes a genetic change (i.e., the mutation of a coding sequence), and the development of cancer is progressed by the combination of such genetic and epigenetic changes. In a MLH1 gene as an example, there is the circumstance in which the function of one allele of the MLH1 gene in colorectal cancer cells is lost due to its mutation or deletion, and the remaining one allele does not function due to promoter methylation. In addition, if the function of MLH1, which is a DNA restoring gene, is lost due to promoter methylation, the occurrence of mutation in other important genes is facilitated to promote the development of cancer.

Most cancers show three common characteristics with respect to CpG, namely, hypermethylation of the promoter CpG islands of tumor-suppressor genes, hypomethylation of the remaining CpG base sites, and an increase in the activity of methylation enzyme, namely, DNA cytosine methyltransferase (DNMT) (Singal, R. & Ginder, G. D., Blood, 93:4059, 1999; Robertson, K. et al., Carcinogensis, 21:461, 2000; Malik, K. & Brown, K. W., Brit. J. Cancer, 83:1583, 2000).

When promoter CpG islands are methylated, the reason why the expression of the corresponding genes is blocked is not clearly established, but is presumed to be because a methyl CpG-binding protein (MECP) or a methyl CpG-binding domain protein (MBD), and histone deacetylase, bind to methylated cytosine, thereby causing a change in the chromatin structure of chromosomes and a change in histone protein.

It is unsettled whether the methylation of promoter CpG islands directly causes the development of cancer or is a secondary change after the development of cancer. However, it is clear that the promoter methylation of tumor-related genes is an important index to cancer, and thus can be used in many applications, including the diagnosis and early detection of cancer, the prediction of the risk of the development of cancer, the prognosis of cancer, follow-up examination after treatment, and the prediction of a response to anticancer therapy. Recently, an attempt to examine the promoter methylation of tumor-related genes in blood, sputum, saliva, feces or urine and to use the examined results for the diagnosis and treatment of various cancers, has been actively conducted (Esteller, M. et al., Cancer Res., 59:67, 1999; Sanchez-Cespedez, M. et al., Cancer Res., 60:892, 2000; Ahlquist, D. A. et al., Gastroenterol., 119:1219, 2000).

In order to maximize the accuracy of cancer diagnosis using promoter methylation, analyze the development of cancer according to each stage and discriminate a change according to cancer and aging, an examination that can accurately analyze the methylation of all the cytosine bases of promoter CpG islands is required. Currently, a standard method for this examination is a bisulfite genome-sequencing method, in which a sample DNA is treated with sodium bisulfite, and all regions of the CpG islands of a target gene to be examined is amplified by PCR, and then, the base sequence of the amplified regions is analyzed. However, this examination has the problem that there are limitations to the number of genes or samples that can be examined at a given time. Other problems are that automation is difficult, and much time and expense are required.

In the Johns Hopkins School of Medicine, the MD Anderson Cancer Center, Charité-Universitatsmedizin Berlin, etc., studies on promoter methylation of cancer-related genes have been actively conducted. The fundamental data thus obtained are interchanged through the DNA Methylation Society (DMS) and stored in MethDB (http://www.methdb.de). Meanwhile, EpiGenX Pharmaceuticals, Inc. is now developing therapeutic agents associated with the methylation of CpG islands, and Epigenomics, Inc. is now conducting studies to apply promoter methylation to cancer diagnosis by examining the promoter methylation using various techniques, such as DNA chips and MALDI-TOF.

Accordingly, the present inventors have made extensive efforts to develop an effective colon-cancer-specific methylation marker which makes it possible to diagnose cancer and the risk of carcinogenesis at an early stage and predict cancer prognosis. As a result, the present inventors have found that GPM6A (NM_005277, Glycoprotein M6A) gene is methylated specifically in colorectal cancer cells and that colorectal cancer can be diagnosed by measuring the degree of methylation using this gene as a biomarker, thereby completing the present disclosure.

DISCLOSURE OF INVENTION

It is a main object of the present disclosure to provide a colorectal cancer-specific methylation biomarker, which is methylated specifically in colorectal cancer cells and can be effectively used for diagnosis of colorectal cancer.

Another object of the present disclosure is to provide a method for detecting colorectal cancer, the method comprising identifying the degree of methylation of the biomarker.

Still another object of the present disclosure is to provide a nucleic acid chip for diagnosing colorectal cancer, which comprises a probe capable of hybridizing with a fragment comprising the CpG island of the colorectal cancer-specific methylation biomarker.

To achieve the above objects, the present disclosure provides a biomarker for diagnosing colorectal cancer, which comprises either the methylated CpG island of the promoter of GPM6A (NM_201591, glycoprotein M6A) gene or the methylated CpG island of a first intron of GPM6A (NM_201591, glycoprotein M6A) gene.

The present disclosure also provides a method for detecting the methylation of a biomarker for colorectal cancer diagnosis, the method comprising the steps of:

(a) isolating DNAs from a clinical sample;

(b) detecting the methylation of the CpG island of the promoter of GPM6A (NM_201591, glycoprotein M6A) gene or the CpG island of a first intron of GPM6A (NM_201591, glycoprotein M6A) gene from the isolated DNAs.

The present disclosure also provides a method for detecting CpG methylation of GPM6A (glycoprotein M6A) gene, the method comprising the steps of:

(a) isolating genomic DNA from a clinical sample;

(b) treating the genomic DNA or a fragment thereof with bisulfite;

(c) amplifying a methylated CpG of GPM6A gene in the bisulfite-treated genomic DNA or fragment thereof from step (b) by using primer(s) to amplify a methylated CpG of the bisulfite-treated GPM6A gene; and

(d) determining whether the CpG of GPM6A was methylated based on whether the DNA was amplified in step (c).

The present disclosure also provides a nucleic acid chip for diagnosing colorectal cancer, which comprises a probe capable of hybridizing with a fragment comprising either the CpG island of the promoter of GPM6A (NM_201591, glycoprotein M6A) gene or the CpG island of a first intron of GPM6A (NM_201591, glycoprotein M6A) gene.

The present disclosure also provides a kit for diagnosing colorectal cancer, which contains: a PCR primer pair for amplifying a fragment comprising the methylated CpG island of a first intron of GPM6A (NM_201591, glycoprotein M6A) gene; and a sequencing primer for pyrosequencing a PCR product amplified by the primer pair.

The present disclosure also provides a kit for detecting CpG methylation of GPM6A (glycoprotein M6A) gene, comprising primer(s) to amplify a methylated CpG of the GPM6A gene.

Other features and embodiments of the present disclosure will be more apparent from the following detailed descriptions and the appended claims

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing a process of discovering a methylation biomarker for colorectal cancer diagnosis from the tissue cells of a normal person and a colorectal cancer patient by a CpG microassay.

FIG. 2 is a schematic diagram showing a process of screening colorectal cancer-specific hypermethylated genes from the CpG microarray data of colorectal cancer.

FIG. 3 is a graphic diagram showing the results of measuring the degree of methylation of 3 biomarker candidate genes in a colorectal cancer cell line and the colon tissues of normal persons by pyrosequencing.

FIG. 4 is a graphic diagram showing the results of measuring the degrees of methylation of GPM6A methylation biomarker in colorectal cancer tissue and adjacent normal tissue by pyrosequencing, and the results of measuring the sensitivity and specificity of GPM6A methylation biomarker for colorectal cancer by ROC curve analysis.

FIG. 5 shows the results of verifying the methylation of a GPM6A biomarker gene in the fecal tissues of normal persons and colorectal cancer patients by methylation-specific PCR.

BEST MODE FOR CARRYING OUT THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Generally, the nomenclature used herein is well known and are commonly employed in the art.

In one aspect, the present disclosure is directed to a biomarker for diagnosing colorectal cancer, which comprises either the methylated CpG island of the promoter of GPM6A (NM_201591, glycoprotein M6A) gene or the methylated CpG island of a first intron of GPM6A (NM_201591, glycoprotein M6A) gene.

In the present disclosure, the CpG island may be located in the intron region of the gene. Herein, the intron region of the GPM6A gene may be located between +501 and +1200 nucleotides (nt) from the transcription start site and may comprise a nucleotide sequence of SEQ ID NO: 1.

In another aspect, the present disclosure is directed to a method for detecting the methylation of a biomarker for colorectal cancer diagnosis, the method comprising the steps of:

(a) isolating DNA from a clinical sample;

(b) detecting the methylation of the CpG island of the promoter of GPM6A (NM_201591, glycoprotein M6A) gene or the CpG island of a first intron of GPM6A (NM_201591, glycoprotein M6A) gene from the isolated DNA.

In the present disclosure, step (b) of detecting the methylation of the CpG island of a first intron of GPM6A (NM_201591, glycoprotein M6A) gene may be performed by detecting the methylation of the region shown by SEQ ID NO: 1.

In the present disclosure, step (b) may be performed by detecting the methylation based on the presence/absense or a change in the base sequence of product amplified by using primers capable of amplifying a fragment comprising the CpG island of the first intron of GPM6A (NM_201591, glycoprotein M6A) gene.

In the present disclosure, step (b) may be performed by a method selected from the group consisting of PCR, methylation-specific PCR, real-time methylation-specific PCR, PCR assay using a methylation DNA-specific binding protein, quantitative PCR, DNA chip-based assay, pyrosequencing, and bisulfate sequencing.

In the present disclosure, the clinical sample may be selected from the group consisting of a tissue, cell, blood, blood plasma, feces, and urine from a patient suspected of cancer or a subject to be diagnosed.

In the present disclosure, 4 biomarker candidate genes showing the greatest difference in the degree of methylation between normal persons and colorectal cancer patients were screened, and among these genes, SDC2, SIM1 and SORCS3 genes were confirmed for diagnosis of colorectal cancer. A method for screening methylation marker genes according to the present disclosure comprises the steps of: (a) isolating genomic DNAs from transformed cells and non-transformed cells; (b) reacting the isolated genomic DNAs with a methylated DNA-binding protein, thereby isolating methylated DNAs; and (c) amplifying the methylated DNAs, hybridizing the amplified DNAs to a CpG microarray, and then selecting genes showing the greatest difference in the degree of methylation between the normal cells and the cancer cells, thereby ensuring methylation marker genes.

The above method for screening biomarker genes can find genes which are differentially methylated in colorectal cancer as well as at various dysplasic stages of the tissue that progresses to colorectal cancer. The screened genes can be used for colorectal cancer screening, risk-assessment, prognosis, disease identification, the diagnosis of disease stages, and the selection of therapeutic targets.

The identification of genes that are methylated in colorectal cancer and abnormalities at various stages of colorectal cancer makes it possible to diagnose colorectal cancer at an early stage in an accurate and effective manner and allows methylation profiling of multiple genes and the identification of new targets for therapeutic intervention. Furthermore, the methylation data according to the present disclosure may be combined with other non-methylation related biomarker detection methods to obtain a more accurate system for colorectal cancer diagnosis.

According to the method of the present disclosure, the progression of colorectal cancer at various stages or phases can be diagnosed by determining the methylation stage of one or more nucleic acid biomarkers obtained from a sample. By comparing the methylation stage of a nucleic acid isolated from a sample at each stage of colorectal cancer with the methylation stage of one or more nucleic acids isolated from a sample in which there is no cell proliferative disorder of colon tissue, a specific stage of colorectal cancer in the sample can be detected. Herein, the methylation stage may be hypermethylation.

In one embodiment of the present disclosure, nucleic acid may be methylated in the regulatory region of a gene. In another embodiment, a gene which is involved in cell transformation can be diagnosed by detecting methylation outside of the regulatory region of the gene, because methylation proceeds inwards from the outside of the gene.

In yet another embodiment of the present disclosure, cells that are likely to form colorectal cancer can be diagnosed at an early stage using the methylation marker genes. When genes confirmed to be methylated in cancer cells are methylated in cells that appear normal clinically or morphologically, this indicates that the normally appearing cells progress to cancer. Thus, colorectal cancer can be diagnosed at an early stage by detecting the methylation of colorectal cancer-specific genes in cells that appear normal.

The use of the methylation marker gene of the present disclosure allows for detection of a cellular proliferative disorder (dysplasia) of colon tissue in a sample. The detection method comprises bringing a sample comprising at least one nucleic acid isolated from a subject into contact with at least one agent capable of determining the methylation state of the nucleic acid. The method comprises detecting the methylation of at least one region in at least one nucleic acid, wherein the methylation of the nucleic acid differs from the methylation state of the same region of a nucleic acid present in a sample in which there is no abnormal growth (dysplastic progression) of colon cells.

In yet another embodiment of the present disclosure, the likelihood of progression of tissue to colorectal cancer can be evaluated by examining the frequency of the methylation of a gene which is specifically methylated in colorectal cancer, and determining the methylation frequency of tissue that is likely to progress to colorectal cancer.

Thus, in still another aspect, the present disclosure is directed to a method for detecting the methylation of colorectal cancer-specific methylation marker gene for colorectal cancer diagnosis, the method comprising the steps of:

(a) preparing a clinical sample containing DNA; and

(b) detecting the methylation of the CpG island of a first intron of GPM6A (NM_005277, glycoprotein M6A) gene in the DNA of the clinical sample.

In the present disclosure, step (b) may be performed by detecting the methylation of the CpG island in the intron region of the gene. Herein, the intron region of the GPM6A gene may be located between +501 and +1200 nucleotides (nt) from the transcription start site and may comprise a nucleotide sequence of SEQ ID NO: 1.

In the present disclosure, step (b) may be performed by a method selected from the group consisting of PCR, methylation-specific PCR, real-time methylation-specific PCR, PCR assay using a methylation DNA-specific binding protein, quantitative PCR, DNA chip-based assay, pyrosequencing, and bisulfate sequencing. In addition, the clinical sample may be selected from the group consisting of a tissue, cell, blood, blood plasma, feces, and urine from a patient suspected of cancer or a subject to be diagnosed, but is not limited thereto.

In one embodiment of the present disclosure, the method for detecting the methylation of a gene may comprise: (a) preparing a clinical sample containing DNA; (b) isolating DNA from the clinical sample; (c) amplifying the isolated DNA using primers capable of amplifying a fragment comprising the CpG island of a first intron of GPM6A (NM_005277, glycoprotein M6A) gene; and (d) determining whether the intron was methylated based on whether the DNA was amplified in step (c).

In yet another aspect, the present disclosure is directed to a nucleic acid chip for diagnosing colorectal cancer, which comprises a probe capable of hybridizing with a fragment comprising the CpG island of a first intron of GPM6A (NM_201591, glycoprotein M6A) gene.

In the present disclosure, the CpG island may be located in the intron region of the gene. Herein, the intron region of the GPM6A gene may be located between +501 and +1200 nucleotides (nt) from the transcription start site and may comprise a nucleotide sequence of SEQ ID NO: 1.

In a further another aspect, the present disclosure is directed to a kit for diagnosing colorectal cancer, which contains: a PCR primer pair for amplifying a fragment comprising the methylated CpG island of a first intron of GPM6A (NM_201591, glycoprotein M6A) gene; and a sequencing primer for pyrosequencing a PCR product amplified by the primer pair.

In the present disclosure, the PCR primer pair may be a primer pair comprising base sequences shown by SEQ ID NOS: 16 and 17 or SEQ ID NO: 18 and 19, and the sequencing primer may comprise a base sequence shown by SEQ ID NO: 15.

In yet another embodiment of the present disclosure, the abnormal growth (dysplasia) of colorectal tissue cells in a sample can be diagnosed by detecting the methylation state of CpG island of a first intron of GPM6A (NM_201591, glycoprotein M6A) gene using a kit.

In the present disclosure, the probe may be selected from the group consisting of the base sequences shown by SEQ ID NOS: 2 to 6, and specific examples thereof are as follows.

The probe capable of hybridizing with the CpG island of a first intron of GPM6A:

1) (SEQ ID NO: 2) gtatttggga aataaagaaa  2) (SEQ ID NO: 3) gactaagaga cccaggatcc gaatagcgag 3) (SEQ ID NO: 4) gttcccacgt tttcatgttc tctttgggga gcaagttgaa  4) (SEQ ID NO: 5) ggcgtccaca ctggctcggg tcactggacg gtggagttcg gcgcagttca 5) (SEQ ID NO: 6) agtttccagg cagggtccgc ttattcggtg cttagcggag gcagcttgga atagctccag 

The use of the diagnostic kit or nucleic acid chip of the present disclosure makes it possible to determine the abnormal growth (dysplastic progression) of colon tissue cells in a sample. The method comprises determining the methylation state of at least one nucleic acid isolated from a sample, wherein the methylation state of the at least one nucleic acid is compared with the methylation stage of a nucleic acid isolated from a sample in which there is no abnormal growth (dysplastic progression) of colorectal cells.

In another embodiment of the present disclosure, transformed colorectal cancer cells can be detected by examining the methylation of the marker gene using said nucleic acid chip.

In still another embodiment of the present disclosure, colorectal cancer can be diagnosed by examining the methylation of the marker gene using said nucleic acid chip.

In yet another embodiment of the present disclosure, the likelihood of progression to colorectal cancer can be diagnosed by examining the methylation of the marker gene in a sample showing a normal phenotype using said kit or nucleic acid chip. The sample that is used in the present disclosure may be solid or liquid tissue, cells, feces, urine, serum, or blood plasma.

Major terms which are used herein are defined as follows.

As used herein, the term “cell transformation” refers to the change in characteristics of a cell from one form to another form such as from normal to abnormal, non-tumorous to tumorous, undifferentiated to differentiated, stem cell to non-stem cell. In addition, the transformation can be recognized by the morphology, phenotype, biochemical characteristics and the like of a cell.

As used herein, the term “early detection” of cancer refers to discovering the likelihood of cancer prior to metastasis, and preferably before observation of a morphological change in a tissue or cell. Furthermore, the term “early detection” of cell transformation refers to the high probability of a cell to undergo transformation in its early stages before the cell is morphologically designated as being transformed.

As used herein, the term “hypermethylation” refers to the methylation of a CpG island.

As used herein, the term “sample” or “clinical sample” is referred to in its broadest sense, and includes any biological sample obtained from an individual, body fluid, a cell line, a tissue culture, depending on the type of assay that is to be performed. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. A tissue biopsy of the colon is a preferred source.

Biomarker for Colorectal Cancer—Use of Cancer Cells for Comparison with Normal Cells

In the present disclosure, “normal” cells refer to those that do not show any abnormal morphological or cytological changes. “Tumor” cells are cancer cells. “Non-tumor” cells are those cells that are part of the diseased tissue but are not considered to be the tumor portion.

In one aspect, the present disclosure is based on the discovery of the relationship between colorectal cancer and the hypermethylation of GPM6A (NM_005277, glycoprotein M6A) gene.

In another embodiment of the present disclosure, a cellular proliferative disorder of colorectal tissue cell can be diagnosed at an early stage by determining the methylation stage of at least one nucleic acid from a subject using the kit or nucleic acid chip of the present disclosure. Herein, the methylation stage of the at least one nucleic acid may be compared with the methylation state of at least one nucleic acid isolated from a subject not having a cellular proliferative disorder of colon tissue. The nucleic acid is preferably a CpG-containing nucleic acid such as a CpG island.

In another embodiment of the present disclosure, a cellular proliferative disorder of colon tissue can be diagnosed by determining the methylation of at least one nucleic acid from a subject using the kit or nucleic acid chip of the present disclosure. Herein, the nucleic acid may be a CpG island gene of GPM6A (NM_005277, glycoprotein M6A) gene. In this embodiment, the methylation of the at least one nucleic acid may be compared with the methylation state of at least one nucleic acid isolated from a subject having no predisposition to a cellular proliferative disorder of colon tissue.

As used herein, “predisposition” refers to the property of being susceptible to a cellular proliferative disorder. A subject having a predisposition to a cellular proliferative disorder has no cellular proliferative disorder, but is a subject having an increased likelihood of having a cellular proliferative disorder.

In another aspect, the present disclosure provides a method for diagnosing a cellular proliferative disorder of colon tissue, the method comprising brining a sample comprising a nucleic acid into contact with an agent capable of determining the methylation state of the sample, and determining the methylation of at least one region of the at least one nucleic acid. Herein, the methylation of the at least one region in the at least one nucleic acid differs from the methylation stage of the same region in a nucleic acid present in a subject in which there is no abnormal growth of cells.

The method of the present disclosure comprises a step of determining the methylation of at least one region of at least one nucleic acid isolated from a subject.

The term “nucleic acid” or “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, or fragments thereof, or single-stranded or double-stranded DNA or RNA of genomic or synthetic origin, sense- or antisense-strand DNA or RNA of genomic or synthetic origin, peptide nucleic acid (PNA), or any DNA-like or RNA-like material of natural or synthetic origin. It will apparent to those of skill in the art that, when the nucleic acid is RNA, the deoxynucleotides A, G, C, and T are replaced by the ribonucleotides A, G, C, and U, respectively.

Any nucleic acid may be used in the present disclosure, given the presence of differently methylated CpG islands can be detected therein. The CpG island is a CpG-rich region in a nucleic acid sequence.

Methylation

In the present disclosure, any nucleic acid sample, in purified or nonpurified form, can be used, provided it contains or is suspected of containing a nucleic acid sequence containing a target locus (e.g., CpG-containing nucleic acid). One nucleic acid region capable of being differentially methylated is a CpG island, a sequence of nucleic acid with an increased density relative to other nucleic acid regions of the dinucleotide CpG. The CpG doublet occurs in vertebrate DNA at only about 20% of the frequency that would be expected from the proportion of G*C base pairs. In certain regions, the density of CpG doublets reaches the predicted value; it is increased by ten-fold relative to the rest of the genome. CpG islands have an average G*C content of about 60%, compared with the 40% average in bulk DNA. The islands take the form of stretches of DNA typically about one to two kilobases long. There are about 45,000 islands in the human genome.

In many genes, the CpG islands begin just upstream of a promoter and extend downstream into the transcribed region. Methylation of a CpG island at a promoter usually suppresses expression of the gene. The islands can also surround the 5′ region of the coding region of the gene as well as the 3′ region of the coding region. Thus, CpG islands can be found in multiple regions of a nucleic acid sequence including upstream of coding sequences in a regulatory region including a promoter region, in the coding regions (e.g., exons), downstream of coding regions in, for example, enhancer regions, and in introns.

Typically, the CpG-containing nucleic acid is DNA. However, the inventive method may employ, for example, samples that contain DNA, or DNA and RNA containing mRNA, wherein DNA or RNA may be single-stranded or double-stranded, or a DNA-RNA hybrid may be included in the sample.

A mixture of nucleic acids may also be used. The specific nucleic acid sequence to be detected may be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be studied be present initially in a pure form; the nucleic acid may be a minor fraction of a complex mixture, such as contained in whole human DNA. Nucleic acids contained in a sample used for detection of methylated CpG islands may be extracted by a variety of techniques such as that described by Sambrook, et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989).

Nucleic acids isolated from a subject are obtained in a biological sample from the subject. If it is desired to detect colorectal cancer or stages of colorectal cancer progression, the nucleic acid may be isolated from colon tissue by scraping or biopsy. Such samples may be obtained by various medical procedures known to those of skill in the art.

In one aspect of the invention, the state of methylation in nucleic acids of the sample obtained from a subject is hypermethylation compared with the same regions of the nucleic acid in a subject not having a cellular proliferative disorder of colon tissue. Hypermethylation as used herein refers to the presence of methylated alleles in one or more nucleic acids. Nucleic acids from a subject not having a cellular proliferative disorder of colon tissue contain no detectable methylated alleles when the same nucleic acids are examined.

Method for Detection of Methylation

Methylation-Specific PCR

When genomic DNA is treated with bisulfite, cytosine in the 5′-CpG′-3 region remains intact, if it was methylated, but the cytosine changes to uracil, if it was unmethylated. Accordingly, based on the base sequence converted after bisulfite treatment, PCR primer sets corresponding to a region having the 5′-CpG-3′ base sequence are constructed. Herein, the constructed primer sets are two kinds of primer sets: a primer set corresponding to the methylated base sequence, and a primer set corresponding to the unmethylated base sequence. When genomic DNA is converted with bisulfite and then amplified by PCR using the above two kinds of primer sets, the PCR product is detected in the PCR mixture employing the primers corresponding to the methylated base sequence, if the genomic DNA was methylated, but the genomic DNA is detected in the PCR mixture employing the primers corresponding to the unmethylated, if the genomic DNA was unmethylated. This methylation can be quantitatively analyzed by agarose gel electrophoresis.

Real-Time Methylation Specific PCR

Real-time methylation-specific PCR is a real-time measurement method modified from the methylation-specific PCR method and comprises treating genomic DNA with bisulfite, designing PCR primers corresponding to the methylated base sequence, and performing real-time PCR using the primers. Methods of detecting the methylation of the genomic DNA include two methods: a method of detection using a TanMan probe complementary to the amplified base sequence; and a method of detection using Sybergreen. Thus, the real-time methylation-specific PCR allows selective quantitative analysis of methylated DNA. Herein, a standard curve is plotted using an in vitro methylated DNA sample, and a gene containing no 5′-CpG-3′ sequence in the base sequence is also amplified as a negative control group for standardization to quantitatively analyze the degree of methylation.

Primer(s) that could amplify a methylated CpG of GPM6A might be used, and primer(s) comprises at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of GPM6A. Specifically, the primer(s) for amplifying a methylated CpG of GPM6A comprise sequence(s) having a homology of 50% or more, specifically at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, with one or more sequence(s) selected from the group consisting of SEQ ID NOs: 24-25, 27-106, 108-189, 191-392, 394-535, 537-658, 660-761, 763-844, 846-967, 969-1070, 1072-1193, 1195-1276, 1278-1359, and 1361-1408.

If required, probe(s) capable of hybridizing with a methylated CpG of GPM6A might be used. The probe(s) capable of hybridizing with a methylated CpG of GPM6A comprise at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of GPM6A. Specifically, probe(s) might comprise sequence(s) having a homology of 50% or more, specifically at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, with one or more sequence(s) selected from the group consisting of SEQ ID NOs: 26, 107, 190, 393, 536, 659, 762, 845, 968, 1071, 1194, 1277 and 1360.

Pyrosequencing

The pyrosequencing method is a quantitative real-time sequencing method modified from the bisulfite sequencing method. Similarly to bisulfite sequencing, genomic DNA is converted by bisulfite treatment, and then, PCR primers corresponding to a region containing no 5′-CpG-3′ base sequence are constructed. Specifically, the genomic DNA is treated with bisulfite, amplified using the PCR primers, and then subjected to real-time base sequence analysis using a sequencing primer. The degree of methylation is expressed as a methylation index by analyzing the amounts of cytosine and thymine in the 5′-CpG-3′ region.

PCR Using Methylated DNA-Specific Binding Protein, Quantitative PCR, and DNA Chip Assay

When a protein binding specifically only to methylated DNA is mixed with DNA, the protein binds specifically only to the methylated DNA. Thus, either PCR using a methylation-specific binding protein or a DNA chip assay allows selective isolation of only methylated DNA. Genomic DNA is mixed with a methylation-specific binding protein, and then only methylated DNA was selectively isolated. The isolated DNA is amplified using PCR primers corresponding to the promoter region, and then methylation of the DNA is measured by agarose gel electrophoresis.

In addition, methylation of DNA can also be measured by a quantitative PCR method, and methylated DNA isolated with a methylated DNA-specific binding protein can be labeled with a fluorescent probe and hybridized to a DNA chip containing complementary probes, thereby measuring methylation of the DNA. Herein, the methylated DNA-specific binding protein may be, but not limited to, McrBt.

Detection of Differential Methylation-Methylation-Sensitive Restriction Endonuclease

Detection of differential methylation can be accomplished by bringing a nucleic acid sample into contact with a methylation-sensitive restriction endonuclease that cleaves only unmethylated CpG sites.

In a separate reaction, the sample is further brought into contact with an isoschizomer of the methylation-sensitive restriction enzyme that cleaves both methylated and unmethylated CpG-sites, thereby cleaving the methylated nucleic acid.

Specific primers are added to the nucleic acid sample, and the nucleic acid is amplified by any conventional method. The presence of an amplified product in the sample treated with the methylation-sensitive restriction enzyme but absence of an amplified product in the sample treated with the isoschizomer of the methylation-sensitive restriction enzyme indicates that methylation has occurred at the nucleic acid region assayed. However, the absence of an amplified product in the sample treated with the methylation-sensitive restriction enzyme together with the absence of an amplified product in the sample treated with the isoschizomer of the methylation-sensitive restriction enzyme indicates that no methylation has occurred at the nucleic acid region assayed.

As used herein, the term “methylation-sensitive restriction enzyme” refers to a restriction enzyme (e.g., SmaI) that includes CG as part of its recognition site and has activity when the C is methylated as compared to when the C is not methylated. Non-limiting examples of methylation-sensitive restriction enzymes include MspI, HpaII, BssHII, BstUI and Nod. Such enzymes can be used alone or in combination. Examples of other methylation-sensitive restriction enzymes include, but are not limited to SacII and EagI.

The isoschizomer of the methylation-sensitive restriction enzyme is a restriction enzyme that recognizes the same recognition site as the methylation-sensitive restriction enzyme but cleaves both methylated and unmethylated CGs. An example thereof includes MspI.

Primers of the present disclosure are designed to be “substantially” complementary to each strand of the locus to be amplified and include the appropriate G or C nucleotides as discussed above. This means that the primers must be sufficiently complementary to hybridize with their respective strands under polymerization reaction conditions. Primers of the present disclosure are used in the amplification process, which is an enzymatic chain reaction (e.g., PCR) in which that a target locus exponentially increases through a number of reaction steps. Typically, one primer is homologous with the negative (−) strand of the locus (antisense primer), and the other primer is homologous with the positive (+) strand (sense primer). After the primers have been annealed to denatured nucleic acid, the nucleic acid chain is extended by an enzyme such as DNA Polymerase I (Klenow), and reactants such as nucleotides, and, as a result, + and − strands containing the target locus sequence are newly synthesized. When the newly synthesized target locus is used as a template and subjected to repeated cycles of denaturing, primer annealing, and extension, exponential synthesis of the target locus sequence occurs. The resulting reaction product is a discrete nucleic acid duplex with termini corresponding to the ends of specific primers employed.

The amplification reaction is PCR which is commonly used in the art. However, alternative methods such as real-time PCR or linear amplification using isothermal enzyme may also be used. In addition, multiplex amplification reactions may also be used.

Detection of Differential Methylation—Bisulfite Sequencing Method

Another method for detecting a methylated CpG-containing nucleic acid comprises the steps of: bringing a nucleic acid-containing sample into contact with an agent that modifies unmethylated cytosine; and amplifying the CpG-containing nucleic acid in the sample using CpG-specific oligonucleotide primers, wherein the oligonucleotide primers distinguish between modified methylated nucleic acid and non-methylated nucleic acid and detect the methylated nucleic acid. The amplification step is optional and desirable, but not essential. The method relies on the PCR reaction to distinguish between modified (e.g., chemically modified) methylated DNA and unmethylated DNA. Such methods are described in U.S. Pat. No. 5,786,146 relating to bisulfite sequencing for detection of methylated nucleic acid.

Kit

The present disclosure provides a kit useful for the detection of a cellular proliferative disorder in a subject.

The present disclosure provides a kit useful for detecting CpG methylation of GPM6A (glycoprotein M6A) gene, comprising primer(s) to amplify a methylated CpG of the GPM6A gene.

Primer(s) that could amplify a methylated CpG of GPM6A might be used, and primer(s) comprises at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of GPM6A. Specifically, the primer(s) for amplifying a methylated CpG of GPM6A comprise sequence(s) having a homology of 50% or more, specifically at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, with one or more sequence(s) selected from the group consisting of SEQ ID NOs: 24-25, 27-106, 108-189, 191-392, 394-535, 537-658, 660-761, 763-844, 846-967, 969-1070, 1072-1193, 1195-1276, 1278-1359, and 1361-1408.

If required, probe(s) capable of hybridizing with a methylated CpG of GPM6A might be used. The probe(s) capable of hybridizing with a methylated CpG of GPM6A comprise at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of GPM6A. Specifically, probe(s) might comprise sequence(s) having a homology of 50% or more, specifically at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, with one or more sequence(s) selected from the group consisting of SEQ ID NOs: 26, 107, 190, 393, 536, 659, 762, 845, 968, 1071, 1194, 1277 and 1360.

The kit of the present disclosure comprises a carrier means compartmentalized to receive a sample therein, one or more containers comprising a second container containing PCR primers for amplification of a 5′-CpG-3′ base sequence, and a third container containing a sequencing primer for pyrosequencing an amplified PCR product.

Carrier means are suited for containing one or more containers such as vials, tubes, and the like, each of the containers comprising one of the separate elements to be used in the method. In view of the description provided herein of the inventive method, those of skill in the art can readily determine the apportionment of the necessary reagents among the containers.

Substrates

After the target nucleic acid region has been amplified, the nucleic acid amplification product can be hybridized to a known gene probe attached to a solid support (substrate) to detect the presence of the nucleic acid sequence.

As used herein, the term “substrate”, when used in reference to a substance, structure, surface or material, means a composition comprising a nonbiological, synthetic, nonliving, planar or round surface that is not heretofore known to comprise a specific binding, hybridization or catalytic recognition site or a plurality of different recognition sites or a number of different recognition sites which exceeds the number of different molecular species comprising the surface, structure or material. Examples of the substrate include, but are not limited to, semiconductors, synthetic (organic) metals, synthetic semiconductors, insulators and dopants; metals, alloys, elements, compounds and minerals; synthetic, cleaved, etched, lithographed, printed, machined and microfabricated slides, devices, structures and surfaces; industrial polymers, plastics, membranes silicon, silicates, glass, metals and ceramics; and wood, paper, cardboard, cotton, wool, cloth, woven and nonwoven fibers, materials and fabrics; and amphibious surfaces.

It is known in the art that several types of membranes have adhesion to nucleic acid sequences. Specific non-limiting examples of these membranes include nitrocellulose or other membranes used for detection of gene expression such as polyvinylchloride, diazotized paper and other commercially available membranes such as GENESCREEN™, ZETAPROBE™ (Biorad) and NYTRAN™. Beads, glass, wafer and metal substrates are also included. Methods for attaching nucleic acids to these objects are well known in the art. Alternatively, screening can be done in a liquid phase.

Hybridization Conditions

In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC/AT content), and nucleic acid type (e.g., RNA/DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.

An example of progressively higher stringency conditions is as follows: 2×SSC/0.1% SDS at room temperature (hybridization conditions); 0.2×SSC/0.1% SDS at room temperature (low stringency conditions); 0.2×SSC/0.1% SDS at 42° C. (moderate stringency conditions); and 0.1×SSC at about 68° C. (high stringency conditions). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary depending on the particular hybridization reaction involved, and can be determined empirically. In general, conditions of high stringency are used for the hybridization of the probe of interest.

Label

The probe of interest can be detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme. Appropriate labeling with such probes is widely known in the art and can be performed by any conventional method.

Examples

Hereinafter, the present disclosure will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples are illustrative purposes only and are not to be construed to limit the scope of the present disclosure.

Example 1: Discovery of Colorectal Cancer-Specific Methylated Genes

In order to screen biomarkers which are methylated specifically in colorectal cancer, 500 ng of each of genomic DNAs from 2 normal persons and genomic DNAs from the cancer tissue and adjacent normal tissue from 12 colorectal cancer patients was sonicated (Vibra Cell, SONICS), thus constructing about 200-300-bp-genomic DNA fragments.

To obtain only methylated DNA from the genomic DNA, a methyl binding domain (Methyl binding domain; MBD) (Fraga et al., Nucleic Acid Res., 31: 1765, 2003) known to bind to methylated DNA was used. Specifically, 2 μg of 6×His-tagged MBD2bt was pre-incubated with 500 ng of the genomic DNA of E. coli JM110 (No. 2638, Biological Resource Center, Korea Research Institute of Bioscience & Biotechnology), and then bound to Ni-NTA magnetic beads (Qiagen, USA). 500 ng of each of the sonicated genomic DNAs isolated from the normal persons and the colorectal cancer patient patients was allowed to react with the beads in the presence of binding buffer solution (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 3 mM MgCl₂, 0.1% Triton-X100, 5% glycerol, 25 mg/ml BSA) at 4° C. for 20 minutes. Then, the beads were washed three times with 500 μL of a binding buffer solution containing 700 mM NaCl, and then methylated DNA bound to the MBD2bt was isolated using the QiaQuick PCR purification kit (Qiagen, USA).

Then, the methylated DNAs bound to the MBD2bt were amplified using a genomic DNA amplification kit (Sigma, USA, Cat. No. WGA2), and 4 μg of the amplified DNAs were labeled with Cy5 using a BioPrime Total Genomic Labeling system I (Invitrogen Corp., USA). To indirectly compare the degree of methylation between the normal person and the colorectal cancer patient, a reference DNA was constructed. Herein, the reference DNA was constructed by mixing the genomic DNAs from the 12 colorectal cancer patients with each other in the same amount, amplifying the genomic DNA mixture using a genomic DNA amplification kit (Sigma, USA, Cat. No. WGA2), and labeling 4 μg of the amplified genomic DNA with Cy3 using a BioPrime Total Genomic Labeling system I (Invitrogen Corp., USA). The reference DNA was mixed with each of the DNAs of the normal persons and the colorectal cancer patients, and then hybridized to 244K human CpG microarrays (Agilent, USA) (see FIG. 1). After the hybridization, the DNA mixture was subjected to a series of washing processes, and then scanned using an Agilent scanner. The calculation of signal values from the microarray images was performed by calculating the relative difference in signal strength between the normal person sample and the colorectal cancer patient sample using Feature Extraction program v. 9.5.3.1 (Agilent).

In order to screen probes having reliable hybridization signals, 64,325 probes having a Cy3 signal value of more than 112.8 in at least 21 arrays among a total of 26 arrays were screened by the cross gene error model using GeneSpring 7.3 program (Agilent, USA). In order to screen probes hypermethylated specifically in colorectal cancer from the above probes, the normally appearing tissue adjacent to the colorectal cancer tissue and the colorectal cancer tissue are compared with each other, and in order to screen probes showing differential methylation, the ANOVA test was performed, thereby screening 3,242 probes (p<0.01). From these probes, 705 probes hypermethylated in the colorectal cancer tissue were further screened, and from these probes, 3 biomarker gene candidates (SLITRK5, ZNF312, GPM6A) showing hypermethylation in two or more adjacent probes present within a distance of about 400 bp were selected (see FIG. 2).

The 4 biomarker candidate genes analyzed using the above analysis method were listed in Table 1 below. In addition, the nucleotide sequence corresponding to the probe of each of the 4 genes showing hypermethylation in the CpG microarray was analyzed using MethPrimer (http://itsa.ucsf.edu/˜urolab/methprimer/indexl.html), thereby confirming CpG islands in the probes.

TABLE 1 List of methylation biomarker candidate genes for colorectal cancer diagnosis Candidate Probe GenBank genes locations ^(a) No. Description SLITRK5 +1,811, +2,046 NM_015567 SLIT and NTRK-like family, member 5 ZNF312 +2,558, +2,646 NM_018008 zinc finger protein 312 GPM6A +554, +786 NM_005277 Glycoprotein M6A ^(a) base pairs (bp) from the transcription start site (+1)

Example 2: Measurement of Methylation of Biomarker Genes in Cancer Cell Lines

In order to additionally confirm the methylation state of the biomarker candidate genes selected in Example 1, pyrosequencing for the promoter and intron region of each gene was performed.

In order to modify unmethylated cytosine to uracil using bisulfite, total genomic DNA was isolated from each of the colorectal cancer cell lines Caco-2 (KCLB No. 30037.1) and HCT116 (KCLB No. 10247), and 200 ng of the genomic DNA was treated with bisulfite using the EZ DNA methylation-gold kit (Zymo Research, USA). When the DNA was treated with bisulfite, unmethylated cytosine was modified to uracil, and the methylated cytosine remained without changes. The DNA treated with bisulfite was eluted in 20 μl of sterile distilled water and subjected to pyrosequencing.

PCR and sequencing primers for performing pyrosequencing for the 3 genes were designed using PSQ assay design program (Biotage, USA). The PCR and sequencing primers for measuring the methylation of each gene are shown in Tables 2 and 3 below.

TABLE 2 PCR primers SEQ ID Size of  Genes Primers Sequences (5′3′)^(a) NOS CpG location^(b) amplicon (bp) SLITRK5 forward TGTTGATTTTTGGTGTA  7 +1949, +1960 253 TTGA +1963, +1989 SLITRK5 reverse AACACATCAACRTCCT  8 +1949, +1960 253 AATTACATA +1963, +1989 ZNF312 forward TGTTTGGTGTAGGGGG  9 +2521, +2527 224 AAGT +2535, +2546 ZNF312 reverse CCCRAAAAAATTATTTT 10 +2521, +2527 224 ACCTCCA +2535, +2546 GPM6A forward GGGAAATAAAGAAAGA 11 +560, +567, 121 TTAAGAGA +572, +598 GPM6A reverse ACCCCRTTTCAACTTAC 12 +560, +567, 121 TC +572, +598 ^(a)Y = C or T; R = A or G ^(b)distances (nucleotides) from the transcription start site (+1): the positions of CpG regions on the genomic DNA used in the measurement of methylation

TABLE 3 Sequences of sequencing primers for methylation marker genes Genes Sequences (5′→3′)^(a) SEQ ID NOS SLITRK5 ATTTTAGTGGTTTAAAGATG 13 ZNF312 TGGGTGTATTGAGAGATTT 14 GPM6A AAGATTAAGAGATTTAGGAT 15 ^(a)Y = C or T; R = A or G 20 ng of the genomic DNA treated with bisulfite was amplified by PCR. In the PCR amplification, a PCR reaction solution (20 ng of the genomic DNA treated with bisulfite, 5 μl of 10×PCR buffer (Enzynomics, Korea), 5 units of Taq polymerase (Enzynomics, Korea), 4 μl of 2.5 mM dNTP (Solgent, Korea), and 2 μl (10 pmole/μl) of PCR primers) was used, and the PCR reaction was performed under the following conditions: predenaturation at 95° C. for 5 min, and then 45 cycles of denaturation at 95° C. for 40 sec, annealing at 60° C. for 45 sec and extension at 72° C. for 40 sec, followed by final extension at 72° C. for 5 min. The amplification of the PCR product was confirmed by electrophoresis on 2.0% agarose gel.

The amplified PCR product was treated with PyroGold reagents (Biotage, USA), and then subjected to pyrosequencing using the PSQ96MA system (Biotage, USA). After the pyrosequencing, the methylation degree of the DNA was measured by calculating the methylation index. The methylation index was calculated by determining the average rate of cytosine binding to each CpG island.

As described above, the degrees of methylation of the biomarker candidate genes in the colorectal cancer cell lines were measured using the pyrosequencing method. As a result, as can be seen in FIG. 3A, the 3 marker genes were all methylated at high levels of 50% in at least one of the cell lines. The 3 genes showed high levels of methylation in the colorectal cancer cell lines, suggesting that these genes are useful as biomarkers for colorectal cancer diagnosis. In order to verify whether these genes are used as biomarkers, the following test was additionally performed using a tissue sample.

Example 3: Measurement of Methylation of Biomarker Candidate Genes in Colon Tissue of Normal Persons

In order for the 3 biomarker candidate gene to have utility as biomarkers for colorectal cancer diagnosis, these genes should show low levels of methylation in the colon tissue of normal persons other than patients, but should show high levels of methylation in colorectal cancer tissue.

To verify whether these genes satisfy these requirements, genomic DNA was isolated from two normal person's colorectal tissues (Biochain) using the QIAamp DNA mini-kit (QIAGEN, USA), and 200 ng of the isolated genomic DNA was treated with bisulfite using the EZ DNA methylation-gold kit (Zymo Research, USA). The treated DNA was eluted in 20 μl of sterile distilled water and subjected to pyrosequencing.

20 ng of the genomic DNA treated with bisulfite was amplified by PCR. In the PCR amplification, a PCR reaction solution (20 ng of the genomic DNA treated with bisulfite, 5 μl of 10×PCR buffer (Enzynomics, Korea), 5 units of Taq polymerase (Enzynomics, Korea), 4 μl of 2.5 mM dNTP (Solgent, Korea), and 2 μl (10 pmole/μl) of PCR primers) was used, and the PCR reaction was performed under the following conditions: predenaturation at 95° C. for 5 min, and then 45 cycles of denaturation at 95° C. for 40 sec, annealing at 60° C. for 45 sec and extension at 72° C. for 40 sec, followed by final extension at 72° C. for 5 min. The amplification of the PCR product was confirmed by electrophoresis on 2.0% agarose gel.

The amplified PCR product was treated with PyroGold reagents (Biotage, USA), and then subjected to pyrosequencing using the PSQ96MA system (Biotage, USA). After the pyrosequencing, the methylation degree of the DNA was measured by calculating the methylation index thereof. The methylation index was calculated by determining the average rate of cytosine binding to each CpG region.

As a result, as can be seen in FIG. 3B, the GPM6A gene among the 3 genes showed the lowest methylation level in the normal tissue. Thus, in order to verify whether the GPM6A gene is useful as a biomarker, the following test was performed using the tissue of colorectal cancer patients.

Example 4: Measurement of Methylation of Biomarker Genes in Tissue of Colorectal Cancer Patients

In order to verify whether the GPM6A gene showing low level of methylation in the colon tissue of normal persons is useful as a biomarker for colorectal cancer diagnosis, genomic DNAs were isolated from colorectal cancer tissues isolated from 96 colorectal cancer patients (the Biochip Research Center in Yonsei University, appointed by the Korean Ministry of Health and Welfare) and the normally appearing tissues adjacent thereto.

200 ng of each of the isolated genomic DNAs was treated with bisulfite using the EZ DNA methylation-gold kit (Zymo Research, USA). Each of the treated DNAs was eluted in 20 μl of sterile distilled water and subjected to pyrosequening.

20 ng of the genomic DNA treated with bisulfite was amplified by PCR. In the PCR amplification, a PCR reaction solution (20 ng of the genomic DNA treated with bisulfite, 5 μl of 10×PCR buffer (Enzynomics, Korea), 5 units of Taq polymerase (Enzynomics, Korea), 4 μl of 2.5 mM dNTP (Solgent, Korea), and 2 μl (10 pmole/μl) of PCR primers) was used, and the PCR reaction solution was performed under the following conditions: predenaturation at 95° C. for 5 min, and then 45 cycles of denaturation at 95° C. for 40 sec, annealing at 60° C. for 45 sec and extension at 72° C. for 40 sec, followed by final extension at 72° C. for 5 min. The amplification of the PCR product was confirmed by electrophoresis on 2.0% agarose gel.

The amplified PCR product was treated with PyroGold reagents (Biotage, USA), and then subjected to pyrosequencing using the PSQ96MA system. After the pyrosequencing, the methylation degree of the DNA was measured by calculating the methylation index thereof. The methylation index was calculated by determining the average rate of cytosine binding to each CpG region.

The degree of metylation of the GPM6A gene was measured. As a result, as can be seen in FIG. 4A, the GPM6A gene showed higher levels of methylation in the colorectal cancer tissues of 72 patents (80.2%) of the 96 patients compared to those in the normally appearing tissues. Table 4 below shows the average values of the methylation levels of the GPM6A biomarker gene in the colorectal cancer tissues and the normally appearing tissues adjacent thereto. In order to confirm whether the level of methylation of the genes statistically significantly differs between the colorectal cancer tissue and the normally appearing tissue, the Chi-Square test was performed. As a result, it could be seen that all the three genes showed statistically significant levels (p<0.01) (see Table 4).

TABLE 4 Results of quantitative analysis of methylation of GPM6A biomarker Average methylation level (%, average ± standard deviation) Normally appearing tissues Colorectal cancer tissues P values ^(a) 6.8 ± 5.7 30.3 ± 19.6 <0.0001 ^(a) p values obtained through the Chi-Square test

Example 5: Evaluation of the Ability of GPM6A Biomarker to Diagnose Colorectal Cancer

For the GPM6A gene confirmed to be useful as colorectal cancer markers in Example 4, receiver operating characteristic (ROC) analysis was performed using MedCalc program (MEDCALC, Belgium) in order to evaluate the ability of the genes to diagnose colorectal cancer.

As a result, as shown in FIG. 4B, the sensitivity and specificity of the GPM6A gene for colorectal cancer were, respectively, 80.2% and 94.8%. This suggests that the GPM6A gene has a very excellent ability to diagnose colorectal cancer. Table 5 shows the results of ROC curve analysis of the GPM6A gene for colorectal cancer diagnosis.

TABLE 5 Results of ROC curve analysis for colorectal cancer diagnosis of the GPM6A methylation biomarker gene AUC (95% C.I)  0.884 (0.830-0.926) Cut-off^(a) >10.31 p 

0.0001 Sensitivity (%)(95% C.I) 80.2 (70.8-87.6) Specificity (%)(95% C.I) 94.8 (88.3-98.3) ^(a)methylation index critera for distinction between normal and cancer samples

Additionally, the GPM6A gene was evaluated for its ability to diagnose colorectal cancer in a fecal sample.

Specifically, measurement of the methylation was performed on two kinds of colorectal cancer cell lines using a nested methylation-specific PCR (MSP) technique, and it was confirmed that all the two kinds of colorectal cancer cell lines were methylated as shown in FIG. 5A. This result is identical to the result of pyrosequencing performed in the above Example 2. Genomic DNAs were isolated from the fecal samples of 4 normal persons and 8 colorectal cancer patients (the Biochip Research Center in Yonsei University, appointed by the Korean Ministry of Health and Welfare). 4 μg of each of the isolated genomic DNAs was treated with bisulfite using the EZ DNA methylation-gold kit (Zymo Research, USA). Each of the treated DNAs was eluted in 20 μl of sterile distilled water and subjected to a nested MSP test. The primer sequences used in the nested MSP test are shown in Table 6 below.

TABLE 6 Primer sequences used in MSP test of GPM6A gene Size of amplified SEQ ID Methylation Primers Primer sequences (5′→3′) product (bp) NOS Methylation Outer-F TTAAAAGGGCGTTTATATTGGTT 233 16 CG Outer-R CCTCGCTCTTCGAAATAACTCGT   17 A Inner-F TAGGGTTCGTTTATTCGGTGTTT 156 18 AGC Inner-R CCTCGCTCTTCGAAATAACTCGT   19 A Non-methylation Outer-F TAAAAGGGTGTTTATATTGGTTT 233 20 GG Outer-R CCCTCACTCTTCAAAATAACTCA   21 TA Inner-F GGTAGGGTTTGTTTATTTGGTGT 160 22 TTAGTG Inner-R TCCCTCACTCTTCAAAATAACTC   23 ATA

1 μg of the genomic DNA treated with bisulfite was amplified by PCR. In the PCR amplification, a PCR reaction solution (20 μg of the genomic DNA treated with bisulfite, 5 μl of 10×PCR buffer (Enzynomics, Korea), 5 units of Taq polymerase (Enzynomics, Korea), 4 μl of 2.5 mM Dntp (Solgent, Korea), and 2 μl (10 pmole/μl) of PCR primers) was used, and the PCR reaction was performed under the following conditions: predenaturation at 95° C. for 5 min, and then 30 cycles of denaturation at 95° C. for 40 sec, annealing at 60° C. for 45 sec and extension at 72° C. for 40 sec, followed by final extension at 72° C. for 5 min. ½ of the PCR product was taken and amplified by PCR for 45 cycles in the same manner as above. The amplification of the PCR products was confirmed by electrophoresis on 2.0% agarose gel.

As a result, as shown in FIG. 5B, it was observed that the GPM6A gene was not methylated in the tissues of the 4 normal persons, but was methylated in 5 (62.5%) of the 8 colorectal cancer patients. This suggests that the GPM6A gene is useful for the diagnosis of colorectal cancer in feces.

Example 6: Evaluation of the Ability of GPM6A Gene to Diagnose Colorectal Cancer by Using qMSP

In order to analyze the ability of GPM6A gene to diagnose colorectal cancer, 686 sets of primers and probes, which could amplify whole CpG island of GPM6A gene and detect methylation specific sites, were designed (Table 7) and methylation specific real time PCR (qMSP) was performed. To achieve the purpose, genome DNA was isolated by using cancer tissues and normal tissues adjacent to cancer tissues from 20 colorectal cancer patients. Treating bisulfite to the above isolated genome DNA (2.0 ug) by using EZ DNA methylation-Gold kit (Zymo Research, USA), and the DNA was subjected to methylation specific real time PCR (qMSP) by eluting with 10 μl distilled water. The qMSP was performed with bisulfite treated genome DNA as a template by using methylation specific primers and probes designed according to Table 7. qMSP used Rotor-Gene Q PCR equipment (Qiagen). Total 20 μl PCR reaction solution (template DNA, 2 μl; 5× AptaTaq DNA Master (Roche Diagnostics), 4 μl; PCR primers, 2 μl (2 pmole/μl), TaqMan probe, 2 μl (2 pmole/μl); D.W. 10 μl) was prepared. Total 40 times of PCR in which the condition is treated at 95° C. for 5 minutes, at 95° C. for 15 seconds and at annealing temperature (58° C.˜61° C.) for 1 minute were performed. The amplification of the PCR product was confirmed by measuring the Ct (cycling threshold) value. Methylated and non-methylated control DNAs were tested with sample DNA by using EpiTect PCR control DNA set (Qiagen, cat. no. 59695). COL2A1 gene (Kristensen et al., 2008) was used as an internal control. The methylation level of each sample was measured by PMR value and the PMR value was calculated as follows:

PMR=2⁻ ^(ΔΔ) ^(Ct)×100, ΔΔCt=[(Ct _((GPM6A)) −Ct _((COL2A1) sample))]−[(Ct _((GPM6A)) −Ct _((COL2A1)Methyl DNA))]

Sensitivity and specificity for set of respective primers and probes were calculated with ROC curve analysis (MedCalc program, Belgium) by using PMR value of cancer tissues and normal tissues adjacent to cancer tissues (Table 8).

TABLE 7 Sequences of primer and probes for GPM6A gene qMSP Size of amplified Sequence Set Primer Sequence (5′→3′) product (bp) No. 1 F696 GTTTATCGTGTTGG 119 24 R15 AAACATAAAAACGT 25 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 2 F697 TTTATCGTGTTGGG 118 27 R15 AAACATAAAAACGT 28 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 3 F698 TTATCGTGTTGGGG 117 29 R15 AAACATAAAAACGT 30 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 4 F699 TATCGTGTTGGGGG 116 31 R15 AAACATAAAAACGT 32 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 5 F700 ATCGTGTTGGGGGC 115 33 R15 AAACATAAAAACGT 34 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 6 F701 TCGTGTTGGGGGCG 114 35 R15 AAACATAAAAACGT 36 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 7 F702 CGTGTTGGGGGCGG 113 37 R15 AAACATAAAAACGT 38 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 8 F703 GTGTTGGGGGCGGT 112 39 R15 AAACATAAAAACGT 40 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 9 F704 TGTTGGGGGCGGTA 111 41 R15 AAACATAAAAACGT 42 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 10 F705 GTTGGGGGCGGTAT 110 43 R15 AAACATAAAAACGT 44 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 11 F706 TTGGGGGCGGTATT 109 45 R15 AAACATAAAAACGT 46 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 12 F707 TGGGGGCGGTATTT 108 47 R15 AAACATAAAAACGT 48 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 13 F708 GGGGGCGGTATTTG 107 49 R15 AAACATAAAAACGT 50 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 14 F709 GGGGCGGTATTTGG 106 51 R15 AAACATAAAAACGT 52 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 15 F710 GGGCGGTATTTGGG 105 53 R15 AAACATAAAAACGT 54 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 16 F711 GGCGGTATTTGGGA 104 55 R15 AAACATAAAAACGT 56 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 17 F712 GCGGTATTTGGGAA 103 57 R15 AAACATAAAAACGT 58 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 18 F713 CGGTATTTGGGAAA 102 59 R15 AAACATAAAAACGT 60 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 19 F714 GGTATTTGGGAAAT 101 61 R15 AAACATAAAAACGT 62 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 20 F715 GTATTTGGGAAATA 100 63 R15 AAACATAAAAACGT 64 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 21 F716 TATTTGGGAAATAA 99 65 R15 AAACATAAAAACGT 66 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 22 F717 ATTTGGGAAATAAA 98 67 R15 AAACATAAAAACGT 68 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 23 F718 TTTGGGAAATAAAG 97 69 R15 AAACATAAAAACGT 70 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 24 F719 TTGGGAAATAAAGA 96 71 R15 AAACATAAAAACGT 72 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 25 F720 TGGGAAATAAAGAA 95 73 R15 AAACATAAAAACGT 74 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 26 F721 GGGAAATAAAGAAA 94 75 R15 AAACATAAAAACGT 76 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 27 F722 GGAAATAAAGAAAG 93 77 R15 AAACATAAAAACGT 78 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 28 F723 GAAATAAAGAAAGA 92 79 R15 AAACATAAAAACGT 80 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 29 F724 AAATAAAGAAAGAT 91 81 R15 AAACATAAAAACGT 82 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 30 F725 AATAAAGAAAGATT 90 83 R15 AAACATAAAAACGT 84 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 31 F726 ATAAAGAAAGATTA 89 85 R15 AAACATAAAAACGT 86 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 32 F727 TAAAGAAAGATTAA 88 87 R15 AAACATAAAAACGT 88 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 33 F728 AAAGAAAGATTAAG 87 89 R15 AAACATAAAAACGT 90 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 34 F729 AAGAAAGATTAAGA 86 91 R15 AAACATAAAAACGT 92 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 35 F730 AGAAAGATTAAGAG 85 93 R15 AAACATAAAAACGT 94 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 36 F731 GAAAGATTAAGAGA 84 95 R15 AAACATAAAAACGT 96 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 37 F732 AAAGATTAAGAGAT 83 97 R15 AAACATAAAAACGT 98 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 38 F733 AAGATTAAGAGATT 82 99 R15 AAACATAAAAACGT 100 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 39 F734 AGATTAAGAGATTT 81 101 R15 AAACATAAAAACGT 102 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 40 F735 GATTAAGAGATTTA 80 103 R15 AAACATAAAAACGT 104 P15 GGGAGATTTTTGTTTTTTCGAGTTTTT 26 41 F736 ATTAAGAGATTTAG 120 105 R16 TTTCCATTTCTCGT 106 P16 GGGAGTAAGTTGAAACGGGGT 107 42 F737 TTAAGAGATTTAGG 119 108 R16 TTTCCATTTCTCGT 109 P16 GGGAGTAAGTTGAAACGGGGT 107 43 F738 TAAGAGATTTAGGA 118 110 R16 TTTCCATTTCTCGT 111 P16 GGGAGTAAGTTGAAACGGGGT 107 44 F739 AAGAGATTTAGGAT 117 112 R16 TTTCCATTTCTCGT 113 P16 GGGAGTAAGTTGAAACGGGGT 107 45 F740 AGAGATTTAGGATT 116 114 R16 TTTCCATTTCTCGT 115 P16 GGGAGTAAGTTGAAACGGGGT 107 46 F741 GAGATTTAGGATTC 115 116 R16 TTTCCATTTCTCGT 117 P16 GGGAGTAAGTTGAAACGGGGT 107 47 F742 AGATTTAGGATTCG 114 118 R16 TTTCCATTTCTCGT 119 P16 GGGAGTAAGTTGAAACGGGGT 107 48 F743 GATTTAGGATTCGA 113 120 R16 TTTCCATTTCTCGT 121 P16 GGGAGTAAGTTGAAACGGGGT 107 49 F744 ATTTAGGATTCGAA 112 122 R16 TTTCCATTTCTCGT 123 P16 GGGAGTAAGTTGAAACGGGGT 107 50 F745 TTTAGGATTCGAAT 111 124 R16 TTTCCATTTCTCGT 125 P16 GGGAGTAAGTTGAAACGGGGT 107 51 F746 TTAGGATTCGAATA 110 126 R16 TTTCCATTTCTCGT 127 P16 GGGAGTAAGTTGAAACGGGGT 107 52 F747 TAGGATTCGAATAG 109 128 R16 TTTCCATTTCTCGT 129 P16 GGGAGTAAGTTGAAACGGGGT 107 53 F748 AGGATTCGAATAGC 108 130 R16 TTTCCATTTCTCGT 131 P16 GGGAGTAAGTTGAAACGGGGT 107 54 F749 GGATTCGAATAGCG 107 132 R16 TTTCCATTTCTCGT 133 P16 GGGAGTAAGTTGAAACGGGGT 107 55 F750 GATTCGAATAGCGA 106 134 R16 TTTCCATTTCTCGT 135 P16 GGGAGTAAGTTGAAACGGGGT 107 56 F751 ATTCGAATAGCGAG 105 136 R16 TTTCCATTTCTCGT 137 P16 GGGAGTAAGTTGAAACGGGGT 107 57 F752 TTCGAATAGCGAGG 104 138 R16 TTTCCATTTCTCGT 139 P16 GGGAGTAAGTTGAAACGGGGT 107 58 F753 TCGAATAGCGAGGC 103 140 R16 TTTCCATTTCTCGT 141 P16 GGGAGTAAGTTGAAACGGGGT 107 59 F754 CGAATAGCGAGGCG 102 142 R16 TTTCCATTTCTCGT 143 P16 GGGAGTAAGTTGAAACGGGGT 107 60 F755 GAATAGCGAGGCGA 101 144 R16 TTTCCATTTCTCGT 145 P16 GGGAGTAAGTTGAAACGGGGT 107 61 F756 AATAGCGAGGCGAT 100 146 R16 TTTCCATTTCTCGT 147 P16 GGGAGTAAGTTGAAACGGGGT 107 62 F757 ATAGCGAGGCGATT 99 148 R16 TTTCCATTTCTCGT 149 P16 GGGAGTAAGTTGAAACGGGGT 107 63 F758 TAGCGAGGCGATTA 98 150 R16 TTTCCATTTCTCGT 151 P16 GGGAGTAAGTTGAAACGGGGT 107 64 F759 AGCGAGGCGATTAT 97 152 R16 TTTCCATTTCTCGT 153 P16 GGGAGTAAGTTGAAACGGGGT 107 65 F760 GCGAGGCGATTATA 96 154 R16 TTTCCATTTCTCGT 155 P16 GGGAGTAAGTTGAAACGGGGT 107 66 F761 CGAGGCGATTATAG 95 156 R16 TTTCCATTTCTCGT 157 P16 GGGAGTAAGTTGAAACGGGGT 107 67 F762 GAGGCGATTATAGG 94 158 R16 TTTCCATTTCTCGT 159 P16 GGGAGTAAGTTGAAACGGGGT 107 68 F763 AGGCGATTATAGGG 93 160 R16 TTTCCATTTCTCGT 161 P16 GGGAGTAAGTTGAAACGGGGT 107 69 F764 GGCGATTATAGGGA 92 162 R16 TTTCCATTTCTCGT 163 P16 GGGAGTAAGTTGAAACGGGGT 107 70 F765 GCGATTATAGGGAG 91 164 R16 TTTCCATTTCTCGT 165 P16 GGGAGTAAGTTGAAACGGGGT 107 71 F766 CGATTATAGGGAGA 90 166 R16 TTTCCATTTCTCGT 167 P16 GGGAGTAAGTTGAAACGGGGT 107 72 F767 GATTATAGGGAGAT 89 168 R16 TTTCCATTTCTCGT 169 P16 GGGAGTAAGTTGAAACGGGGT 107 73 F768 ATTATAGGGAGATT 88 170 R16 TTTCCATTTCTCGT 171 P16 GGGAGTAAGTTGAAACGGGGT 107 74 F769 TTATAGGGAGATTT 87 172 R16 TTTCCATTTCTCGT 173 P16 GGGAGTAAGTTGAAACGGGGT 107 75 F770 TATAGGGAGATTTT 86 174 R16 TTTCCATTTCTCGT 175 P16 GGGAGTAAGTTGAAACGGGGT 107 76 F771 ATAGGGAGATTTTT 85 176 R16 TTTCCATTTCTCGT 177 P16 GGGAGTAAGTTGAAACGGGGT 107 77 F772 TAGGGAGATTTTTG 84 178 R16 TTTCCATTTCTCGT 179 P16 GGGAGTAAGTTGAAACGGGGT 107 78 F773 AGGGAGATTTTTGT 83 180 R16 TTTCCATTTCTCGT 181 P16 GGGAGTAAGTTGAAACGGGGT 107 79 F774 GGGAGATTTTTGTT 82 182 R16 TTTCCATTTCTCGT 183 P16 GGGAGTAAGTTGAAACGGGGT 107 80 F775 GGAGATTTTTGTTT 81 184 R16 TTTCCATTTCTCGT 185 P16 GGGAGTAAGTTGAAACGGGGT 107 81 F776 GAGATTTTTGTTTT 80 186 R16 TTTCCATTTCTCGT 187 P16 GGGAGTAAGTTGAAACGGGGT 107 82 F777 AGATTTTTGTTTTT 180 188 R17 TAAACACTTCGTTT 189 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 83 F778 GATTTTTGTTTTTT 179 191 R17 TAAACACTTCGTTT 192 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 84 F779 ATTTTTGTTTTTTC 178 193 R17 TAAACACTTCGTTT 194 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 85 F780 TTTTTGTTTTTTCG 177 195 R17 TAAACACTTCGTTT 196 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 86 F781 TTTTGTTTTTTCGA 176 197 R17 TAAACACTTCGTTT 198 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 87 F782 TTTGTTTTTTCGAG 175 199 R17 TAAACACTTCGTTT 200 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 88 F783 TTGTTTTTTCGAGT 174 201 R17 TAAACACTTCGTTT 202 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 89 F784 TGTTTTTTCGAGTT 173 203 R17 TAAACACTTCGTTT 204 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 90 F785 GTTTTTTCGAGTTT 172 205 R17 TAAACACTTCGTTT 206 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 91 F786 TTTTTTCGAGTTTT 171 207 R17 TAAACACTTCGTTT 208 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 92 F787 TTTTTCGAGTTTTT 170 209 R17 TAAACACTTCGTTT 210 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 93 F788 TTTTCGAGTTTTTA 169 211 R17 TAAACACTTCGTTT 212 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 94 F789 TTTCGAGTTTTTAC 168 213 R17 TAAACACTTCGTTT 214 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 95 F790 TTCGAGTTTTTACG 167 215 R17 TAAACACTTCGTTT 216 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 96 F791 TCGAGTTTTTACGT 166 217 R17 TAAACACTTCGTTT 218 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 97 F792 CGAGTTTTTACGTT 165 219 R17 TAAACACTTCGTTT 220 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 98 F793 GAGTTTTTACGTTT 164 221 R17 TAAACACTTCGTTT 222 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 99 F794 AGTTTTTACGTTTT 163 223 R17 TAAACACTTCGTTT 224 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 100 F795 GTTTTTACGTTTTT 162 225 R17 TAAACACTTCGTTT 226 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 101 F796 TTTTTACGTTTTTA 161 227 R17 TAAACACTTCGTTT 228 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 102 F797 TTTTACGTTTTTAT 160 229 R17 TAAACACTTCGTTT 230 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 103 F798 TTTACGTTTTTATG 159 231 R17 TAAACACTTCGTTT 232 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 104 F799 TTACGTTTTTATGT 158 233 R17 TAAACACTTCGTTT 234 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 105 F800 TACGTTTTTATGTT 157 235 R17 TAAACACTTCGTTT 236 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 106 F801 ACGTTTTTATGTTT 156 237 R17 TAAACACTTCGTTT 238 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 107 F802 CGTTTTTATGTTTT 155 239 R17 TAAACACTTCGTTT 240 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 108 F803 GTTTTTATGTTTTT 154 241 R17 TAAACACTTCGTTT 242 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 109 F804 TTTTTATGTTTTTT 153 243 R17 TAAACACTTCGTTT 244 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 110 F805 TTTTATGTTTTTTT 152 245 R17 TAAACACTTCGTTT 246 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 111 F806 TTTATGTTTTTTTT 151 247 R17 TAAACACTTCGTTT 248 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 112 F807 TTATGTTTTTTTTG 150 249 R17 TAAACACTTCGTTT 250 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 113 F808 TATGTTTTTTTTGG 149 251 R17 TAAACACTTCGTTT 252 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 114 F809 ATGTTTTTTTTGGG 148 253 R17 TAAACACTTCGTTT 254 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 115 F810 TGTTTTTTTTGGGG 147 255 R17 TAAACACTTCGTTT 256 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 116 F811 GTTTTTTTTGGGGA 146 257 R17 TAAACACTTCGTTT 258 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 117 F812 TTTTTTTTGGGGAG 145 259 R17 TAAACACTTCGTTT 260 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 118 F813 TTTTTTTGGGGAGT 144 261 R17 TAAACACTTCGTTT 262 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 119 F814 TTTTTTGGGGAGTA 143 263 R17 TAAACACTTCGTTT 264 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 120 F815 TTTTTGGGGAGTAA 142 265 R17 TAAACACTTCGTTT 266 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 121 F816 TTTTGGGGAGTAAG 141 267 R17 TAAACACTTCGTTT 268 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 122 F817 TTTGGGGAGTAAGT 140 269 R17 TAAACACTTCGTTT 270 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 123 F818 TTGGGGAGTAAGTT 139 271 R17 TAAACACTTCGTTT 272 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 124 F819 TGGGGAGTAAGTTG 138 273 R17 TAAACACTTCGTTT 274 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 125 F820 GGGGAGTAAGTTGA 137 275 R17 TAAACACTTCGTTT 276 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 126 F821 GGGAGTAAGTTGAA 136 277 R17 TAAACACTTCGTTT 278 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 127 F822 GGAGTAAGTTGAAA 135 279 R17 TAAACACTTCGTTT 280 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 128 F823 GAGTAAGTTGAAAC 134 281 R17 TAAACACTTCGTTT 282 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 129 F824 AGTAAGTTGAAACG 133 283 R17 TAAACACTTCGTTT 284 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 130 F825 GTAAGTTGAAACGG 132 285 R17 TAAACACTTCGTTT 286 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 131 F826 TAAGTTGAAACGGG 131 287 R17 TAAACACTTCGTTT 288 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 132 F827 AAGTTGAAACGGGG 130 289 R17 TAAACACTTCGTTT 290 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 133 F828 AGTTGAAACGGGGT 129 291 R17 TAAACACTTCGTTT 292 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 134 F829 GTTGAAACGGGGTA 128 293 R17 TAAACACTTCGTTT 294 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 135 F830 TTGAAACGGGGTAC 127 295 R17 TAAACACTTCGTTT 296 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 136 F831 TGAAACGGGGTACG 126 297 R17 TAAACACTTCGTTT 298 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 137 F832 GAAACGGGGTACGA 125 299 R17 TAAACACTTCGTTT 300 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 138 F833 AAACGGGGTACGAG 124 301 R17 TAAACACTTCGTTT 302 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 139 F834 AACGGGGTACGAGA 123 303 R17 TAAACACTTCGTTT 304 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 140 F835 ACGGGGTACGAGAA 122 305 R17 TAAACACTTCGTTT 306 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 141 F836 CGGGGTACGAGAAA 121 307 R17 TAAACACTTCGTTT 308 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 142 F837 GGGGTACGAGAAAT 120 309 R17 TAAACACTTCGTTT 310 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 143 F838 GGGTACGAGAAATG 119 311 R17 TAAACACTTCGTTT 312 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 144 F839 GGTACGAGAAATGG 118 313 R17 TAAACACTTCGTTT 314 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 145 F840 GTACGAGAAATGGA 117 315 R17 TAAACACTTCGTTT 316 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 146 F841 TACGAGAAATGGAA 116 317 R17 TAAACACTTCGTTT 318 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 147 F842 ACGAGAAATGGAAA 115 319 R17 TAAACACTTCGTTT 320 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 148 F843 CGAGAAATGGAAAT 114 321 R17 TAAACACTTCGTTT 322 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 149 F844 GAGAAATGGAAATT 113 323 R17 TAAACACTTCGTTT 324 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 150 F845 AGAAATGGAAATTT 112 325 R17 TAAACACTTCGTTT 326 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 151 F846 GAAATGGAAATTTT 111 327 R17 TAAACACTTCGTTT 328 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 152 F847 AAATGGAAATTTTT 110 329 R17 TAAACACTTCGTTT 330 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 153 F848 AATGGAAATTTTTT 109 331 R17 TAAACACTTCGTTT 332 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 154 F849 ATGGAAATTTTTTA 108 333 R17 TAAACACTTCGTTT 334 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 155 F850 TGGAAATTTTTTAA 107 335 R17 TAAACACTTCGTTT 336 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 156 F851 GGAAATTTTTTAAA 106 337 R17 TAAACACTTCGTTT 338 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 157 F852 GAAATTTTTTAAAA 105 339 R17 TAAACACTTCGTTT 340 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 158 F853 AAATTTTTTAAAAT 104 341 R17 TAAACACTTCGTTT 342 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 159 F854 AATTTTTTAAAATT 103 343 R17 TAAACACTTCGTTT 344 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 160 F855 ATTTTTTAAAATTT 102 345 R17 TAAACACTTCGTTT 346 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 161 F856 TTTTTTAAAATTTT 101 347 R17 TAAACACTTCGTTT 348 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 162 F857 TTTTTAAAATTTTT 100 349 R17 TAAACACTTCGTTT 350 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 163 F858 TTTTAAAATTTTTA 99 351 R17 TAAACACTTCGTTT 352 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 164 F859 TTTAAAATTTTTAT 98 353 R17 TAAACACTTCGTTT 354 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 165 F860 TTAAAATTTTTATT 97 355 R17 TAAACACTTCGTTT 356 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 166 F861 TAAAATTTTTATTT 96 357 R17 TAAACACTTCGTTT 358 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 167 F862 AAAATTTTTATTTT 95 359 R17 TAAACACTTCGTTT 360 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 168 F863 AAATTTTTATTTTG 94 361 R17 TAAACACTTCGTTT 362 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 169 F864 AATTTTTATTTTGT 93 363 R17 TAAACACTTCGTTT 364 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 170 F865 ATTTTTATTTTGTA 92 365 R17 TAAACACTTCGTTT 366 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 171 F866 TTTTTATTTTGTAT 91 367 R17 TAAACACTTCGTTT 368 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 172 F867 TTTTATTTTGTATA 90 369 R17 TAAACACTTCGTTT 370 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 173 F868 TTTATTTTGTATAG 89 371 R17 TAAACACTTCGTTT 372 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 174 F869 TTATTTTGTATAGG 88 373 R17 TAAACACTTCGTTT 374 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 175 F870 TATTTTGTATAGGT 87 375 R17 TAAACACTTCGTTT 376 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 176 F871 ATTTTGTATAGGTT 86 377 R17 TAAACACTTCGTTT 378 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 177 F872 TTTTGTATAGGTTT 85 379 R17 TAAACACTTCGTTT 380 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 178 F873 TTTGTATAGGTTTG 84 381 R17 TAAACACTTCGTTT 382 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 179 F874 TTGTATAGGTTTGA 83 383 R17 TAAACACTTCGTTT 384 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 180 F875 TGTATAGGTTTGAG 82 385 R17 TAAACACTTCGTTT 386 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 181 F876 GTATAGGTTTGAGT 81 387 R17 TAAACACTTCGTTT 388 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 182 F877 TATAGGTTTGAGTA 80 389 R17 TAAACACTTCGTTT 390 P17 TTGGGGATAATTTTTAGTTTTTTTTT 190 183 F878 ATAGGTTTGAGTAG 150 391 R18 TCACGCTATTTACC 392 P18 TTGATTTTTTTTAAATTTTATTTG 393 184 F879 TAGGTTTGAGTAGA 149 394 R18 TCACGCTATTTACC 395 P18 TTGATTTTTTTTAAATTTTATTTG 393 185 F880 AGGTTTGAGTAGAG 148 396 R18 TCACGCTATTTACC 397 P18 TTGATTTTTTTTAAATTTTATTTG 393 186 F881 GGTTTGAGTAGAGG 147 398 R18 TCACGCTATTTACC 399 P18 TTGATTTTTTTTAAATTTTATTTG 393 187 F882 GTTTGAGTAGAGGA 146 400 R18 TCACGCTATTTACC 401 P18 TTGATTTTTTTTAAATTTTATTTG 393 188 F883 TTTGAGTAGAGGAA 145 402 R18 TCACGCTATTTACC 403 P18 TTGATTTTTTTTAAATTTTATTTG 393 189 F884 TTGAGTAGAGGAAG 144 404 R18 TCACGCTATTTACC 405 P18 TTGATTTTTTTTAAATTTTATTTG 393 190 F885 TGAGTAGAGGAAGG 143 406 R18 TCACGCTATTTACC 407 P18 TTGATTTTTTTTAAATTTTATTTG 393 191 F886 GAGTAGAGGAAGGT 142 408 R18 TCACGCTATTTACC 409 P18 TTGATTTTTTTTAAATTTTATTTG 393 192 F887 AGTAGAGGAAGGTG 141 410 R18 TCACGCTATTTACC 411 P18 TTGATTTTTTTTAAATTTTATTTG 393 193 F888 GTAGAGGAAGGTGT 140 412 R18 TCACGCTATTTACC 413 P18 TTGATTTTTTTTAAATTTTATTTG 393 194 F889 TAGAGGAAGGTGTT 139 414 R18 TCACGCTATTTACC 415 P18 TTGATTTTTTTTAAATTTTATTTG 393 195 F890 AGAGGAAGGTGTTG 138 416 R18 TCACGCTATTTACC 417 P18 TTGATTTTTTTTAAATTTTATTTG 393 196 F891 GAGGAAGGTGTTGG 137 418 R18 TCACGCTATTTACC 419 P18 TTGATTTTTTTTAAATTTTATTTG 393 197 F892 AGGAAGGTGTTGGT 136 420 R18 TCACGCTATTTACC 421 P18 TTGATTTTTTTTAAATTTTATTTG 393 198 F893 GGAAGGTGTTGGTG 135 422 R18 TCACGCTATTTACC 423 P18 TTGATTTTTTTTAAATTTTATTTG 393 199 F894 GAAGGTGTTGGTGT 134 424 R18 TCACGCTATTTACC 425 P18 TTGATTTTTTTTAAATTTTATTTG 393 200 F895 AAGGTGTTGGTGTA 133 426 R18 TCACGCTATTTACC 427 P18 TTGATTTTTTTTAAATTTTATTTG 393 201 F896 AGGTGTTGGTGTAG 132 428 R18 TCACGCTATTTACC 429 P18 TTGATTTTTTTTAAATTTTATTTG 393 202 F897 GGTGTTGGTGTAGG 131 430 R18 TCACGCTATTTACC 431 P18 TTGATTTTTTTTAAATTTTATTTG 393 203 F898 GTGTTGGTGTAGGG 130 432 R18 TCACGCTATTTACC 433 P18 TTGATTTTTTTTAAATTTTATTTG 393 204 F899 TGTTGGTGTAGGGT 129 434 R18 TCACGCTATTTACC 435 P18 TTGATTTTTTTTAAATTTTATTTG 393 205 F900 GTTGGTGTAGGGTT 128 436 R18 TCACGCTATTTACC 437 P18 TTGATTTTTTTTAAATTTTATTTG 393 206 F901 TTGGTGTAGGGTTA 127 438 R18 TCACGCTATTTACC 439 P18 TTGATTTTTTTTAAATTTTATTTG 393 207 F902 TGGTGTAGGGTTAG 126 440 R18 TCACGCTATTTACC 441 P18 TTGATTTTTTTTAAATTTTATTTG 393 208 F903 GGTGTAGGGTTAGA 125 442 R18 TCACGCTATTTACC 443 P18 TTGATTTTTTTTAAATTTTATTTG 393 209 F904 GTGTAGGGTTAGAT 124 444 R18 TCACGCTATTTACC 445 P18 TTGATTTTTTTTAAATTTTATTTG 393 210 F905 TGTAGGGTTAGATT 123 446 R18 TCACGCTATTTACC 447 P18 TTGATTTTTTTTAAATTTTATTTG 393 211 F906 GTAGGGTTAGATTG 122 448 R18 TCACGCTATTTACC 449 P18 TTGATTTTTTTTAAATTTTATTTG 393 212 F907 TAGGGTTAGATTGG 121 450 R18 TCACGCTATTTACC 451 P18 TTGATTTTTTTTAAATTTTATTTG 393 213 F908 AGGGTTAGATTGGG 120 452 R18 TCACGCTATTTACC 453 P18 TTGATTTTTTTTAAATTTTATTTG 393 214 F909 GGGTTAGATTGGGG 119 454 R18 TCACGCTATTTACC 455 P18 TTGATTTTTTTTAAATTTTATTTG 393 215 F910 GGTTAGATTGGGGA 118 456 R18 TCACGCTATTTACC 457 P18 TTGATTTTTTTTAAATTTTATTTG 393 216 F911 GTTAGATTGGGGAT 117 458 R18 TCACGCTATTTACC 459 P18 TTGATTTTTTTTAAATTTTATTTG 393 217 F912 TTAGATTGGGGATA 116 460 R18 TCACGCTATTTACC 461 P18 TTGATTTTTTTTAAATTTTATTTG 393 218 F913 TAGATTGGGGATAA 115 462 R18 TCACGCTATTTACC 463 P18 TTGATTTTTTTTAAATTTTATTTG 393 219 F914 AGATTGGGGATAAT 114 464 R18 TCACGCTATTTACC 465 P18 TTGATTTTTTTTAAATTTTATTTG 393 220 F915 GATTGGGGATAATT 113 466 R18 TCACGCTATTTACC 467 P18 TTGATTTTTTTTAAATTTTATTTG 393 221 F916 ATTGGGGATAATTT 112 468 R18 TCACGCTATTTACC 469 P18 TTGATTTTTTTTAAATTTTATTTG 393 222 F917 TTGGGGATAATTTT 111 470 R18 TCACGCTATTTACC 471 P18 TTGATTTTTTTTAAATTTTATTTG 393 223 F918 TGGGGATAATTTTT 110 472 R18 TCACGCTATTTACC 473 P18 TTGATTTTTTTTAAATTTTATTTG 393 224 F919 GGGGATAATTTTTA 109 474 R18 TCACGCTATTTACC 475 P18 TTGATTTTTTTTAAATTTTATTTG 393 225 F920 GGGATAATTTTTAG 108 476 R18 TCACGCTATTTACC 477 P18 TTGATTTTTTTTAAATTTTATTTG 393 226 F921 GGATAATTTTTAGT 107 478 R18 TCACGCTATTTACC 479 P18 TTGATTTTTTTTAAATTTTATTTG 393 227 F922 GATAATTTTTAGTT 106 480 R18 TCACGCTATTTACC 481 P18 TTGATTTTTTTTAAATTTTATTTG 393 228 F923 ATAATTTTTAGTTT 105 482 R18 TCACGCTATTTACC 483 P18 TTGATTTTTTTTAAATTTTATTTG 393 229 F924 TAATTTTTAGTTTT 104 484 R18 TCACGCTATTTACC 485 P18 TTGATTTTTTTTAAATTTTATTTG 393 230 F925 AATTTTTAGTTTTT 103 486 R18 TCACGCTATTTACC 487 P18 TTGATTTTTTTTAAATTTTATTTG 393 231 F926 ATTTTTAGTTTTTT 102 488 R18 TCACGCTATTTACC 489 P18 TTGATTTTTTTTAAATTTTATTTG 393 232 F927 TTTTTAGTTTTTTT 101 490 R18 TCACGCTATTTACC 491 P18 TTGATTTTTTTTAAATTTTATTTG 393 233 F928 TTTTAGTTTTTTTT 100 492 R18 TCACGCTATTTACC 493 P18 TTGATTTTTTTTAAATTTTATTTG 393 234 F929 TTTAGTTTTTTTTT 99 494 R18 TCACGCTATTTACC 495 P18 TTGATTTTTTTTAAATTTTATTTG 393 235 F930 TTAGTTTTTTTTTA 98 496 R18 TCACGCTATTTACC 497 P18 TTGATTTTTTTTAAATTTTATTTG 393 236 F931 TAGTTTTTTTTTAA 97 498 R18 TCACGCTATTTACC 499 P18 TTGATTTTTTTTAAATTTTATTTG 393 237 F932 AGTTTTTTTTTAAA 96 500 R18 TCACGCTATTTACC 501 P18 TTGATTTTTTTTAAATTTTATTTG 393 238 F933 GTTTTTTTTTAAAC 95 502 R18 TCACGCTATTTACC 503 P18 TTGATTTTTTTTAAATTTTATTTG 393 239 F934 TTTTTTTTTAAACG 94 504 R18 TCACGCTATTTACC 505 P18 TTGATTTTTTTTAAATTTTATTTG 393 240 F935 TTTTTTTTAAACGA 93 506 R18 TCACGCTATTTACC 507 P18 TTGATTTTTTTTAAATTTTATTTG 393 241 F936 TTTTTTTAAACGAA 92 508 R18 TCACGCTATTTACC 509 P18 TTGATTTTTTTTAAATTTTATTTG 393 242 F937 TTTTTTAAACGAAG 91 510 R18 TCACGCTATTTACC 511 P18 TTGATTTTTTTTAAATTTTATTTG 393 243 F938 TTTTTAAACGAAGT 90 512 R18 TCACGCTATTTACC 513 P18 TTGATTTTTTTTAAATTTTATTTG 393 244 F939 TTTTAAACGAAGTG 89 514 R18 TCACGCTATTTACC 515 P18 TTGATTTTTTTTAAATTTTATTTG 393 245 F940 TTTAAACGAAGTGT 88 516 R18 TCACGCTATTTACC 517 P18 TTGATTTTTTTTAAATTTTATTTG 393 246 F941 TTAAACGAAGTGTT 87 518 R18 TCACGCTATTTACC 519 P18 TTGATTTTTTTTAAATTTTATTTG 393 247 F942 TAAACGAAGTGTTT 86 520 R18 TCACGCTATTTACC 521 P18 TTGATTTTTTTTAAATTTTATTTG 393 248 F943 AAACGAAGTGTTTA 85 522 R18 TCACGCTATTTACC 523 P18 TTGATTTTTTTTAAATTTTATTTG 393 249 F944 AACGAAGTGTTTAT 84 524 R18 TCACGCTATTTACC 525 P18 TTGATTTTTTTTAAATTTTATTTG 393 250 F945 ACGAAGTGTTTATT 83 526 R18 TCACGCTATTTACC 527 P18 TTGATTTTTTTTAAATTTTATTTG 393 251 F946 CGAAGTGTTTATTT 82 528 R18 TCACGCTATTTACC 529 P18 TTGATTTTTTTTAAATTTTATTTG 393 252 F947 GAAGTGTTTATTTG 81 530 R18 TCACGCTATTTACC 531 P18 TTGATTTTTTTTAAATTTTATTTG 393 253 F948 AAGTGTTTATTTGT 80 532 R18 TCACGCTATTTACC 533 P18 TTGATTTTTTTTAAATTTTATTTG 393 254 F949 AGTGTTTATTTGTA 140 534 R19 AAACTAAACTACGC 535 P19 CGGGTTATTGGACGGTGGAGTTCG 536 255 F950 GTGTTTATTTGTAT 139 537 R19 AAACTAAACTACGC 538 P19 CGGGTTATTGGACGGTGGAGTTCG 536 256 F951 TGTTTATTTGTATA 138 539 R19 AAACTAAACTACGC 540 P19 CGGGTTATTGGACGGTGGAGTTCG 536 257 F952 GTTTATTTGTATAA 137 541 R19 AAACTAAACTACGC 542 P19 CGGGTTATTGGACGGTGGAGTTCG 536 258 F953 TTTATTTGTATAAA 136 543 R19 AAACTAAACTACGC 544 P19 CGGGTTATTGGACGGTGGAGTTCG 536 259 F954 TTATTTGTATAAAA 135 545 R19 AAACTAAACTACGC 546 P19 CGGGTTATTGGACGGTGGAGTTCG 536 260 F955 TATTTGTATAAAAG 134 547 R19 AAACTAAACTACGC 548 P19 CGGGTTATTGGACGGTGGAGTTCG 536 261 F956 ATTTGTATAAAAGG 133 549 R19 AAACTAAACTACGC 550 P19 CGGGTTATTGGACGGTGGAGTTCG 536 262 F957 TTTGTATAAAAGGT 132 551 R19 AAACTAAACTACGC 552 P19 CGGGTTATTGGACGGTGGAGTTCG 536 263 F958 TTGTATAAAAGGTT 131 553 R19 AAACTAAACTACGC 554 P19 CGGGTTATTGGACGGTGGAGTTCG 536 264 F959 TGTATAAAAGGTTT 130 555 R19 AAACTAAACTACGC 556 P19 CGGGTTATTGGACGGTGGAGTTCG 536 265 F960 GTATAAAAGGTTTG 129 557 R19 AAACTAAACTACGC 558 P19 CGGGTTATTGGACGGTGGAGTTCG 536 266 F961 TATAAAAGGTTTGA 128 559 R19 AAACTAAACTACGC 560 P19 CGGGTTATTGGACGGTGGAGTTCG 536 267 F962 ATAAAAGGTTTGAG 127 561 R19 AAACTAAACTACGC 562 P19 CGGGTTATTGGACGGTGGAGTTCG 536 268 F963 TAAAAGGTTTGAGG 126 563 R19 AAACTAAACTACGC 564 P19 CGGGTTATTGGACGGTGGAGTTCG 536 269 F964 AAAAGGTTTGAGGT 125 565 R19 AAACTAAACTACGC 566 P19 CGGGTTATTGGACGGTGGAGTTCG 536 270 F965 AAAGGTTTGAGGTT 124 567 R19 AAACTAAACTACGC 568 P19 CGGGTTATTGGACGGTGGAGTTCG 536 271 F966 AAGGTTTGAGGTTG 123 569 R19 AAACTAAACTACGC 570 P19 CGGGTTATTGGACGGTGGAGTTCG 536 272 F967 AGGTTTGAGGTTGA 122 571 R19 AAACTAAACTACGC 572 P19 CGGGTTATTGGACGGTGGAGTTCG 536 273 F968 GGTTTGAGGTTGAG 121 573 R19 AAACTAAACTACGC 574 P19 CGGGTTATTGGACGGTGGAGTTCG 536 274 F969 GTTTGAGGTTGAGG 120 575 R19 AAACTAAACTACGC 576 P19 CGGGTTATTGGACGGTGGAGTTCG 536 275 F970 TTTGAGGTTGAGGT 119 577 R19 AAACTAAACTACGC 578 P19 CGGGTTATTGGACGGTGGAGTTCG 536 276 F971 TTGAGGTTGAGGTT 118 579 R19 AAACTAAACTACGC 580 P19 CGGGTTATTGGACGGTGGAGTTCG 536 277 F972 TGAGGTTGAGGTTG 117 581 R19 AAACTAAACTACGC 582 P19 CGGGTTATTGGACGGTGGAGTTCG 536 278 F973 GAGGTTGAGGTTGA 116 583 R19 AAACTAAACTACGC 584 P19 CGGGTTATTGGACGGTGGAGTTCG 536 279 F974 AGGTTGAGGTTGAA 115 585 R19 AAACTAAACTACGC 586 P19 CGGGTTATTGGACGGTGGAGTTCG 536 280 F975 GGTTGAGGTTGAAG 114 587 R19 AAACTAAACTACGC 588 P19 CGGGTTATTGGACGGTGGAGTTCG 536 281 F976 GTTGAGGTTGAAGG 113 589 R19 AAACTAAACTACGC 590 P19 CGGGTTATTGGACGGTGGAGTTCG 536 282 F977 TTGAGGTTGAAGGT 112 591 R19 AAACTAAACTACGC 592 P19 CGGGTTATTGGACGGTGGAGTTCG 536 283 F978 TGAGGTTGAAGGTT 111 593 R19 AAACTAAACTACGC 594 P19 CGGGTTATTGGACGGTGGAGTTCG 536 284 F979 GAGGTTGAAGGTTG 110 595 R19 AAACTAAACTACGC 596 P19 CGGGTTATTGGACGGTGGAGTTCG 536 285 F980 AGGTTGAAGGTTGA 109 597 R19 AAACTAAACTACGC 598 P19 CGGGTTATTGGACGGTGGAGTTCG 536 286 F981 GGTTGAAGGTTGAT 108 599 R19 AAACTAAACTACGC 600 P19 CGGGTTATTGGACGGTGGAGTTCG 536 287 F982 GTTGAAGGTTGATT 107 601 R19 AAACTAAACTACGC 602 P19 CGGGTTATTGGACGGTGGAGTTCG 536 288 F983 TTGAAGGTTGATTT 106 603 R19 AAACTAAACTACGC 604 P19 CGGGTTATTGGACGGTGGAGTTCG 536 289 F984 TGAAGGTTGATTTT 105 605 R19 AAACTAAACTACGC 606 P19 CGGGTTATTGGACGGTGGAGTTCG 536 290 F985 GAAGGTTGATTTTT 104 607 R19 AAACTAAACTACGC 608 P19 CGGGTTATTGGACGGTGGAGTTCG 536 291 F986 AAGGTTGATTTTTT 103 609 R19 AAACTAAACTACGC 610 P19 CGGGTTATTGGACGGTGGAGTTCG 536 292 F987 AGGTTGATTTTTTT 102 611 R19 AAACTAAACTACGC 612 P19 CGGGTTATTGGACGGTGGAGTTCG 536 293 F988 GGTTGATTTTTTTT 101 613 R19 AAACTAAACTACGC 614 P19 CGGGTTATTGGACGGTGGAGTTCG 536 294 F989 GTTGATTTTTTTTA 100 615 R19 AAACTAAACTACGC 616 P19 CGGGTTATTGGACGGTGGAGTTCG 536 295 F990 TTGATTTTTTTTAA 99 617 R19 AAACTAAACTACGC 618 P19 CGGGTTATTGGACGGTGGAGTTCG 536 296 F991 TGATTTTTTTTAAA 98 619 R19 AAACTAAACTACGC 620 P19 CGGGTTATTGGACGGTGGAGTTCG 536 297 F992 GATTTTTTTTAAAT 97 621 R19 AAACTAAACTACGC 622 P19 CGGGTTATTGGACGGTGGAGTTCG 536 298 F993 ATTTTTTTTAAATT 96 623 R19 AAACTAAACTACGC 624 P19 CGGGTTATTGGACGGTGGAGTTCG 536 299 F994 TTTTTTTTAAATTT 95 625 R19 AAACTAAACTACGC 626 P19 CGGGTTATTGGACGGTGGAGTTCG 536 300 F995 TTTTTTTAAATTTT 94 627 R19 AAACTAAACTACGC 628 P19 CGGGTTATTGGACGGTGGAGTTCG 536 301 F996 TTTTTTAAATTTTA 93 629 R19 AAACTAAACTACGC 630 P19 CGGGTTATTGGACGGTGGAGTTCG 536 302 F997 TTTTTAAATTTTAT 92 631 R19 AAACTAAACTACGC 632 P19 CGGGTTATTGGACGGTGGAGTTCG 536 303 F998 TTTTAAATTTTATT 91 633 R19 AAACTAAACTACGC 634 P19 CGGGTTATTGGACGGTGGAGTTCG 536 304 F999 TTTAAATTTTATTT 90 635 R19 AAACTAAACTACGC 636 P19 CGGGTTATTGGACGGTGGAGTTCG 536 305 F1000 TTAAATTTTATTTG 89 637 R19 AAACTAAACTACGC 638 P19 CGGGTTATTGGACGGTGGAGTTCG 536 306 F1001 TAAATTTTATTTGG 88 639 R19 AAACTAAACTACGC 640 P19 CGGGTTATTGGACGGTGGAGTTCG 536 307 F1002 AAATTTTATTTGGG 87 641 R19 AAACTAAACTACGC 642 P19 CGGGTTATTGGACGGTGGAGTTCG 536 308 F1003 AATTTTATTTGGGT 86 643 R19 AAACTAAACTACGC 644 P19 CGGGTTATTGGACGGTGGAGTTCG 536 309 F1004 ATTTTATTTGGGTA 85 645 R19 AAACTAAACTACGC 646 P19 CGGGTTATTGGACGGTGGAGTTCG 536 310 F1005 TTTTATTTGGGTAA 84 647 R19 AAACTAAACTACGC 648 P19 CGGGTTATTGGACGGTGGAGTTCG 536 311 F1006 TTTATTTGGGTAAA 83 649 R19 AAACTAAACTACGC 650 P19 CGGGTTATTGGACGGTGGAGTTCG 536 312 F1007 TTATTTGGGTAAAT 82 651 R19 AAACTAAACTACGC 652 P19 CGGGTTATTGGACGGTGGAGTTCG 536 313 F1008 TATTTGGGTAAATA 81 653 R19 AAACTAAACTACGC 654 P19 CGGGTTATTGGACGGTGGAGTTCG 536 314 F1009 ATTTGGGTAAATAG 80 655 R19 AAACTAAACTACGC 656 P19 CGGGTTATTGGACGGTGGAGTTCG 536 315 F1010 TTTGGGTAAATAGC 130 657 R20 ACTACCTCCGCTAA 658 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 316 F1011 TTGGGTAAATAGCG 129 660 R20 ACTACCTCCGCTAA 661 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 317 F1012 TGGGTAAATAGCGT 128 662 R20 ACTACCTCCGCTAA 663 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 318 F1013 GGGTAAATAGCGTG 127 664 R20 ACTACCTCCGCTAA 665 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 319 F1014 GGTAAATAGCGTGA 126 666 R20 ACTACCTCCGCTAA 667 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 320 F1015 GTAAATAGCGTGAT 125 668 R20 ACTACCTCCGCTAA 669 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 321 F1016 TAAATAGCGTGATT 124 670 R20 ACTACCTCCGCTAA 671 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 322 F1017 AAATAGCGTGATTA 123 672 R20 ACTACCTCCGCTAA 673 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 323 F1018 AATAGCGTGATTAA 122 674 R20 ACTACCTCCGCTAA 675 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 324 F1019 ATAGCGTGATTAAA 121 676 R20 ACTACCTCCGCTAA 677 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 325 F1020 TAGCGTGATTAAAA 120 678 R20 ACTACCTCCGCTAA 679 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 326 F1021 AGCGTGATTAAAAG 119 680 R20 ACTACCTCCGCTAA 681 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 327 F1022 GCGTGATTAAAAGG 118 682 R20 ACTACCTCCGCTAA 683 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 328 F1023 CGTGATTAAAAGGG 117 684 R20 ACTACCTCCGCTAA 685 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 329 F1024 GTGATTAAAAGGGC 116 686 R20 ACTACCTCCGCTAA 687 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 330 F1025 TGATTAAAAGGGCG 115 688 R20 ACTACCTCCGCTAA 689 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 331 F1026 GATTAAAAGGGCGT 114 690 R20 ACTACCTCCGCTAA 691 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 332 F1027 ATTAAAAGGGCGTT 113 692 R20 ACTACCTCCGCTAA 693 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 333 F1028 TTAAAAGGGCGTTT 112 694 R20 ACTACCTCCGCTAA 695 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 334 F1029 TAAAAGGGCGTTTA 111 696 R20 ACTACCTCCGCTAA 697 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 335 F1030 AAAAGGGCGTTTAT 110 698 R20 ACTACCTCCGCTAA 699 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 336 F1031 AAAGGGCGTTTATA 109 700 R20 ACTACCTCCGCTAA 701 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 337 F1032 AAGGGCGTTTATAT 108 702 R20 ACTACCTCCGCTAA 703 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 338 F1033 AGGGCGTTTATATT 107 704 R20 ACTACCTCCGCTAA 705 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 339 F1034 GGGCGTTTATATTG 106 706 R20 ACTACCTCCGCTAA 707 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 340 F1035 GGCGTTTATATTGG 105 708 R20 ACTACCTCCGCTAA 709 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 341 F1036 GCGTTTATATTGGT 104 710 R20 ACTACCTCCGCTAA 711 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 342 F1037 CGTTTATATTGGTT 103 712 R20 ACTACCTCCGCTAA 713 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 343 F1038 GTTTATATTGGTTC 102 714 R20 ACTACCTCCGCTAA 715 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 344 F1039 TTTATATTGGTTCG 101 716 R20 ACTACCTCCGCTAA 717 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 345 F1040 TTATATTGGTTCGG 100 718 R20 ACTACCTCCGCTAA 719 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 346 F1041 TATATTGGTTCGGG 99 720 R20 ACTACCTCCGCTAA 721 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 347 F1042 ATATTGGTTCGGGT 98 722 R20 ACTACCTCCGCTAA 723 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 348 F1043 TATTGGTTCGGGTT 97 724 R20 ACTACCTCCGCTAA 725 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 349 F1044 ATTGGTTCGGGTTA 96 726 R20 ACTACCTCCGCTAA 727 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 350 F1045 TTGGTTCGGGTTAT 95 728 R20 ACTACCTCCGCTAA 729 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 351 F1046 TGGTTCGGGTTATT 94 730 R20 ACTACCTCCGCTAA 731 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 352 F1047 GGTTCGGGTTATTG 93 732 R20 ACTACCTCCGCTAA 733 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 353 F1048 GTTCGGGTTATTGG 92 734 R20 ACTACCTCCGCTAA 735 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 354 F1049 TTCGGGTTATTGGA 91 736 R20 ACTACCTCCGCTAA 737 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 355 F1050 TCGGGTTATTGGAC 90 738 R20 ACTACCTCCGCTAA 739 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 356 F1051 CGGGTTATTGGACG 89 740 R20 ACTACCTCCGCTAA 741 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 357 F1052 GGGTTATTGGACGG 88 742 R20 ACTACCTCCGCTAA 743 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 358 F1053 GGTTATTGGACGGT 87 744 R20 ACTACCTCCGCTAA 745 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 359 F1054 GTTATTGGACGGTG 86 746 R20 ACTACCTCCGCTAA 747 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 360 F1055 TTATTGGACGGTGG 85 748 R20 ACTACCTCCGCTAA 749 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 361 F1056 TATTGGACGGTGGA 84 750 R20 ACTACCTCCGCTAA 751 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 362 F1057 ATTGGACGGTGGAG 83 752 R20 ACTACCTCCGCTAA 753 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 363 F1058 TTGGACGGTGGAGT 82 754 R20 ACTACCTCCGCTAA 755 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 364 F1059 TGGACGGTGGAGTT 81 756 R20 ACTACCTCCGCTAA 757 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 365 F1060 GGACGGTGGAGTTC 80 758 R20 ACTACCTCCGCTAA 759 P20 AGGTAGGGTTCGTTTATTCGGTGT 659 366 F1061 GACGGTGGAGTTCG 120 760 R21 CCCCTCCGCCACAC 761 P21 GGAATAGTTTTAGGAATGTGATTGC 762 367 F1062 ACGGTGGAGTTCGG 119 763 R21 CCCCTCCGCCACAC 764 P21 GGAATAGTTTTAGGAATGTGATTGC 762 368 F1063 CGGTGGAGTTCGGC 118 765 R21 CCCCTCCGCCACAC 766 P21 GGAATAGTTTTAGGAATGTGATTGC 762 369 F1064 GGTGGAGTTCGGCG 117 767 R21 CCCCTCCGCCACAC 768 P21 GGAATAGTTTTAGGAATGTGATTGC 762 370 F1065 GTGGAGTTCGGCGT 116 769 R21 CCCCTCCGCCACAC 770 P21 GGAATAGTTTTAGGAATGTGATTGC 762 371 F1066 TGGAGTTCGGCGTA 115 771 R21 CCCCTCCGCCACAC 772 P21 GGAATAGTTTTAGGAATGTGATTGC 762 372 F1067 GGAGTTCGGCGTAG 114 773 R21 CCCCTCCGCCACAC 774 P21 GGAATAGTTTTAGGAATGTGATTGC 762 373 F1068 GAGTTCGGCGTAGT 113 775 R21 CCCCTCCGCCACAC 776 P21 GGAATAGTTTTAGGAATGTGATTGC 762 374 F1069 AGTTCGGCGTAGTT 112 777 R21 CCCCTCCGCCACAC 778 P21 GGAATAGTTTTAGGAATGTGATTGC 762 375 F1070 GTTCGGCGTAGTTT 111 779 R21 CCCCTCCGCCACAC 780 P21 GGAATAGTTTTAGGAATGTGATTGC 762 376 F1071 TTCGGCGTAGTTTA 110 781 R21 CCCCTCCGCCACAC 782 P21 GGAATAGTTTTAGGAATGTGATTGC 762 377 F1072 TCGGCGTAGTTTAG 109 783 R21 CCCCTCCGCCACAC 784 P21 GGAATAGTTTTAGGAATGTGATTGC 762 378 F1073 CGGCGTAGTTTAGT 108 785 R21 CCCCTCCGCCACAC 786 P21 GGAATAGTTTTAGGAATGTGATTGC 762 379 F1074 GGCGTAGTTTAGTT 107 787 R21 CCCCTCCGCCACAC 788 P21 GGAATAGTTTTAGGAATGTGATTGC 762 380 F1075 GCGTAGTTTAGTTT 106 789 R21 CCCCTCCGCCACAC 790 P21 GGAATAGTTTTAGGAATGTGATTGC 762 381 F1076 CGTAGTTTAGTTTC 105 791 R21 CCCCTCCGCCACAC 792 P21 GGAATAGTTTTAGGAATGTGATTGC 762 382 F1077 GTAGTTTAGTTTCG 104 793 R21 CCCCTCCGCCACAC 794 P21 GGAATAGTTTTAGGAATGTGATTGC 762 383 F1078 TAGTTTAGTTTCGT 103 795 R21 CCCCTCCGCCACAC 796 P21 GGAATAGTTTTAGGAATGTGATTGC 762 384 F1079 AGTTTAGTTTCGTT 102 797 R21 CCCCTCCGCCACAC 798 P21 GGAATAGTTTTAGGAATGTGATTGC 762 385 F1080 GTTTAGTTTCGTTT 101 799 R21 CCCCTCCGCCACAC 800 P21 GGAATAGTTTTAGGAATGTGATTGC 762 386 F1081 TTTAGTTTCGTTTA 100 801 R21 CCCCTCCGCCACAC 802 P21 GGAATAGTTTTAGGAATGTGATTGC 762 387 F1082 TTAGTTTCGTTTAA 99 803 R21 CCCCTCCGCCACAC 804 P21 GGAATAGTTTTAGGAATGTGATTGC 762 388 F1083 TAGTTTCGTTTAAG 98 805 R21 CCCCTCCGCCACAC 806 P21 GGAATAGTTTTAGGAATGTGATTGC 762 389 F1084 AGTTTCGTTTAAGT 97 807 R21 CCCCTCCGCCACAC 808 P21 GGAATAGTTTTAGGAATGTGATTGC 762 390 F1085 GTTTCGTTTAAGTT 96 809 R21 CCCCTCCGCCACAC 810 P21 GGAATAGTTTTAGGAATGTGATTGC 762 391 F1086 TTTCGTTTAAGTTT 95 811 R21 CCCCTCCGCCACAC 812 P21 GGAATAGTTTTAGGAATGTGATTGC 762 392 F1087 TTCGTTTAAGTTTT 94 813 R21 CCCCTCCGCCACAC 814 P21 GGAATAGTTTTAGGAATGTGATTGC 762 393 F1088 TCGTTTAAGTTTTT 93 815 R21 CCCCTCCGCCACAC 816 P21 GGAATAGTTTTAGGAATGTGATTGC 762 394 F1089 CGTTTAAGTTTTTA 92 817 R21 CCCCTCCGCCACAC 818 P21 GGAATAGTTTTAGGAATGTGATTGC 762 395 F1090 GTTTAAGTTTTTAG 91 819 R21 CCCCTCCGCCACAC 820 P21 GGAATAGTTTTAGGAATGTGATTGC 762 396 F1091 TTTAAGTTTTTAGG 90 821 R21 CCCCTCCGCCACAC 822 P21 GGAATAGTTTTAGGAATGTGATTGC 762 397 F1092 TTAAGTTTTTAGGT 89 823 R21 CCCCTCCGCCACAC 824 P21 GGAATAGTTTTAGGAATGTGATTGC 762 398 F1093 TAAGTTTTTAGGTA 88 825 R21 CCCCTCCGCCACAC 826 P21 GGAATAGTTTTAGGAATGTGATTGC 762 399 F1094 AAGTTTTTAGGTAG 87 827 R21 CCCCTCCGCCACAC 828 P21 GGAATAGTTTTAGGAATGTGATTGC 762 400 F1095 AGTTTTTAGGTAGG 86 829 R21 CCCCTCCGCCACAC 830 P21 GGAATAGTTTTAGGAATGTGATTGC 762 401 F1096 GTTTTTAGGTAGGG 85 831 R21 CCCCTCCGCCACAC 832 P21 GGAATAGTTTTAGGAATGTGATTGC 762 402 F1097 TTTTTAGGTAGGGT 84 833 R21 CCCCTCCGCCACAC 834 P21 GGAATAGTTTTAGGAATGTGATTGC 762 403 F1098 TTTTAGGTAGGGTT 83 835 R21 CCCCTCCGCCACAC 836 P21 GGAATAGTTTTAGGAATGTGATTGC 762 404 F1099 TTTAGGTAGGGTTC 82 837 R21 CCCCTCCGCCACAC 838 P21 GGAATAGTTTTAGGAATGTGATTGC 762 405 F1100 TTAGGTAGGGTTCG 81 839 R21 CCCCTCCGCCACAC 840 P21 GGAATAGTTTTAGGAATGTGATTGC 762 406 F1101 TAGGTAGGGTTCGT 80 841 R21 CCCCTCCGCCACAC 842 P21 GGAATAGTTTTAGGAATGTGATTGC 762 407 F1102 AGGTAGGGTTCGTT 140 843 R22 TCGTAAAACCCTAC 844 P22 TCGATTTATATTTAGTATTAGGAC 845 408 F1103 GGTAGGGTTCGTTT 139 846 R22 TCGTAAAACCCTAC 847 P22 TCGATTTATATTTAGTATTAGGAC 845 409 F1104 GTAGGGTTCGTTTA 138 848 R22 TCGTAAAACCCTAC 849 P22 TCGATTTATATTTAGTATTAGGAC 845 410 F1105 TAGGGTTCGTTTAT 137 850 R22 TCGTAAAACCCTAC 851 P22 TCGATTTATATTTAGTATTAGGAC 845 411 F1106 AGGGTTCGTTTATT 136 852 R22 TCGTAAAACCCTAC 853 P22 TCGATTTATATTTAGTATTAGGAC 845 412 F1107 GGGTTCGTTTATTC 135 854 R22 TCGTAAAACCCTAC 855 P22 TCGATTTATATTTAGTATTAGGAC 845 413 F1108 GGTTCGTTTATTCG 134 856 R22 TCGTAAAACCCTAC 857 P22 TCGATTTATATTTAGTATTAGGAC 845 414 F1109 GTTCGTTTATTCGG 133 858 R22 TCGTAAAACCCTAC 859 P22 TCGATTTATATTTAGTATTAGGAC 845 415 F1110 TTCGTTTATTCGGT 132 860 R22 TCGTAAAACCCTAC 861 P22 TCGATTTATATTTAGTATTAGGAC 845 416 F1111 TCGTTTATTCGGTG 131 862 R22 TCGTAAAACCCTAC 863 P22 TCGATTTATATTTAGTATTAGGAC 845 417 F1112 CGTTTATTCGGTGT 130 864 R22 TCGTAAAACCCTAC 865 P22 TCGATTTATATTTAGTATTAGGAC 845 418 F1113 GTTTATTCGGTGTT 129 866 R22 TCGTAAAACCCTAC 867 P22 TCGATTTATATTTAGTATTAGGAC 845 419 F1114 TTTATTCGGTGTTT 128 868 R22 TCGTAAAACCCTAC 869 P22 TCGATTTATATTTAGTATTAGGAC 845 420 F1115 TTATTCGGTGTTTA 127 870 R22 TCGTAAAACCCTAC 871 P22 TCGATTTATATTTAGTATTAGGAC 845 421 F1116 TATTCGGTGTTTAG 126 872 R22 TCGTAAAACCCTAC 873 P22 TCGATTTATATTTAGTATTAGGAC 845 422 F1117 ATTCGGTGTTTAGC 125 874 R22 TCGTAAAACCCTAC 875 P22 TCGATTTATATTTAGTATTAGGAC 845 423 F1118 TTCGGTGTTTAGCG 124 876 R22 TCGTAAAACCCTAC 877 P22 TCGATTTATATTTAGTATTAGGAC 845 424 F1119 TCGGTGTTTAGCGG 123 878 R22 TCGTAAAACCCTAC 879 P22 TCGATTTATATTTAGTATTAGGAC 845 425 F1120 CGGTGTTTAGCGGA 122 880 R22 TCGTAAAACCCTAC 881 P22 TCGATTTATATTTAGTATTAGGAC 845 426 F1121 GGTGTTTAGCGGAG 121 882 R22 TCGTAAAACCCTAC 883 P22 TCGATTTATATTTAGTATTAGGAC 845 427 F1122 GTGTTTAGCGGAGG 120 884 R22 TCGTAAAACCCTAC 885 P22 TCGATTTATATTTAGTATTAGGAC 845 428 F1123 TGTTTAGCGGAGGT 119 886 R22 TCGTAAAACCCTAC 887 P22 TCGATTTATATTTAGTATTAGGAC 845 429 F1124 GTTTAGCGGAGGTA 118 888 R22 TCGTAAAACCCTAC 889 P22 TCGATTTATATTTAGTATTAGGAC 845 430 F1125 TTTAGCGGAGGTAG 117 890 R22 TCGTAAAACCCTAC 891 P22 TCGATTTATATTTAGTATTAGGAC 845 431 F1126 TTAGCGGAGGTAGT 116 892 R22 TCGTAAAACCCTAC 893 P22 TCGATTTATATTTAGTATTAGGAC 845 432 F1127 TAGCGGAGGTAGTT 115 894 R22 TCGTAAAACCCTAC 895 P22 TCGATTTATATTTAGTATTAGGAC 845 433 F1128 AGCGGAGGTAGTTT 114 896 R22 TCGTAAAACCCTAC 897 P22 TCGATTTATATTTAGTATTAGGAC 845 434 F1129 GCGGAGGTAGTTTG 113 898 R22 TCGTAAAACCCTAC 899 P22 TCGATTTATATTTAGTATTAGGAC 845 435 F1130 CGGAGGTAGTTTGG 112 900 R22 TCGTAAAACCCTAC 901 P22 TCGATTTATATTTAGTATTAGGAC 845 436 F1131 GGAGGTAGTTTGGA 111 902 R22 TCGTAAAACCCTAC 903 P22 TCGATTTATATTTAGTATTAGGAC 845 437 F1132 GAGGTAGTTTGGAA 110 904 R22 TCGTAAAACCCTAC 905 P22 TCGATTTATATTTAGTATTAGGAC 845 438 F1133 AGGTAGTTTGGAAT 109 906 R22 TCGTAAAACCCTAC 907 P22 TCGATTTATATTTAGTATTAGGAC 845 439 F1134 GGTAGTTTGGAATA 108 908 R22 TCGTAAAACCCTAC 909 P22 TCGATTTATATTTAGTATTAGGAC 845 440 F1135 GTAGTTTGGAATAG 107 910 R22 TCGTAAAACCCTAC 911 P22 TCGATTTATATTTAGTATTAGGAC 845 441 F1136 TAGTTTGGAATAGT 106 912 R22 TCGTAAAACCCTAC 913 P22 TCGATTTATATTTAGTATTAGGAC 845 442 F1137 AGTTTGGAATAGTT 105 914 R22 TCGTAAAACCCTAC 915 P22 TCGATTTATATTTAGTATTAGGAC 845 443 F1138 GTTTGGAATAGTTT 104 916 R22 TCGTAAAACCCTAC 917 P22 TCGATTTATATTTAGTATTAGGAC 845 444 F1139 TTTGGAATAGTTTT 103 918 R22 TCGTAAAACCCTAC 919 P22 TCGATTTATATTTAGTATTAGGAC 845 445 F1140 TTGGAATAGTTTTA 102 920 R22 TCGTAAAACCCTAC 921 P22 TCGATTTATATTTAGTATTAGGAC 845 446 F1141 TGGAATAGTTTTAG 101 922 R22 TCGTAAAACCCTAC 923 P22 TCGATTTATATTTAGTATTAGGAC 845 447 F1142 GGAATAGTTTTAGG 100 924 R22 TCGTAAAACCCTAC 925 P22 TCGATTTATATTTAGTATTAGGAC 845 448 F1143 GAATAGTTTTAGGA 99 926 R22 TCGTAAAACCCTAC 927 P22 TCGATTTATATTTAGTATTAGGAC 845 449 F1144 AATAGTTTTAGGAA 98 928 R22 TCGTAAAACCCTAC 929 P22 TCGATTTATATTTAGTATTAGGAC 845 450 F1145 ATAGTTTTAGGAAT 97 930 R22 TCGTAAAACCCTAC 931 P22 TCGATTTATATTTAGTATTAGGAC 845 451 F1146 TAGTTTTAGGAATG 96 932 R22 TCGTAAAACCCTAC 933 P22 TCGATTTATATTTAGTATTAGGAC 845 452 F1147 AGTTTTAGGAATGT 95 934 R22 TCGTAAAACCCTAC 935 P22 TCGATTTATATTTAGTATTAGGAC 845 453 F1148 GTTTTAGGAATGTG 94 936 R22 TCGTAAAACCCTAC 937 P22 TCGATTTATATTTAGTATTAGGAC 845 454 F1149 TTTTAGGAATGTGA 93 938 R22 TCGTAAAACCCTAC 939 P22 TCGATTTATATTTAGTATTAGGAC 845 455 F1150 TTTAGGAATGTGAT 92 940 R22 TCGTAAAACCCTAC 941 P22 TCGATTTATATTTAGTATTAGGAC 845 456 F1151 TTAGGAATGTGATT 91 942 R22 TCGTAAAACCCTAC 943 P22 TCGATTTATATTTAGTATTAGGAC 845 457 F1152 TAGGAATGTGATTG 90 944 R22 TCGTAAAACCCTAC 945 P22 TCGATTTATATTTAGTATTAGGAC 845 458 F1153 AGGAATGTGATTGC 89 946 R22 TCGTAAAACCCTAC 947 P22 TCGATTTATATTTAGTATTAGGAC 845 459 F1154 GGAATGTGATTGCG 88 948 R22 TCGTAAAACCCTAC 949 P22 TCGATTTATATTTAGTATTAGGAC 845 460 F1155 GAATGTGATTGCGT 87 950 R22 TCGTAAAACCCTAC 951 P22 TCGATTTATATTTAGTATTAGGAC 845 461 F1156 AATGTGATTGCGTG 86 952 R22 TCGTAAAACCCTAC 953 P22 TCGATTTATATTTAGTATTAGGAC 845 462 F1157 ATGTGATTGCGTGT 85 954 R22 TCGTAAAACCCTAC 955 P22 TCGATTTATATTTAGTATTAGGAC 845 463 F1158 TGTGATTGCGTGTG 84 956 R22 TCGTAAAACCCTAC 957 P22 TCGATTTATATTTAGTATTAGGAC 845 464 F1159 GTGATTGCGTGTGG 83 958 R22 TCGTAAAACCCTAC 959 P22 TCGATTTATATTTAGTATTAGGAC 845 465 F1160 TGATTGCGTGTGGC 82 960 R22 TCGTAAAACCCTAC 961 P22 TCGATTTATATTTAGTATTAGGAC 845 466 F1161 GATTGCGTGTGGCG 81 962 R22 TCGTAAAACCCTAC 963 P22 TCGATTTATATTTAGTATTAGGAC 845 467 F1162 ATTGCGTGTGGCGG 80 964 R22 TCGTAAAACCCTAC 965 P22 TCGATTTATATTTAGTATTAGGAC 845 468 F1163 TTGCGTGTGGCGGA 130 966 R23 AATAACGAATACTT 967 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 469 F1164 TGCGTGTGGCGGAG 129 969 R23 AATAACGAATACTT 970 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 470 F1165 GCGTGTGGCGGAGG 128 971 R23 AATAACGAATACTT 972 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 471 F1166 CGTGTGGCGGAGGG 127 973 R23 AATAACGAATACTT 974 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 472 F1167 GTGTGGCGGAGGGG 126 975 R23 AATAACGAATACTT 976 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 473 F1168 TGTGGCGGAGGGGA 125 977 R23 AATAACGAATACTT 978 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 474 F1169 GTGGCGGAGGGGAG 124 979 R23 AATAACGAATACTT 980 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 475 F1170 TGGCGGAGGGGAGG 123 981 R23 AATAACGAATACTT 982 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 476 F1171 GGCGGAGGGGAGGA 122 983 R23 AATAACGAATACTT 984 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 477 F1172 GCGGAGGGGAGGAA 121 985 R23 AATAACGAATACTT 986 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 478 F1173 CGGAGGGGAGGAAG 120 987 R23 AATAACGAATACTT 988 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 479 F1174 GGAGGGGAGGAAGA 119 989 R23 AATAACGAATACTT 990 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 480 F1175 GAGGGGAGGAAGAA 118 991 R23 AATAACGAATACTT 992 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 481 F1176 AGGGGAGGAAGAAT 117 993 R23 AATAACGAATACTT 994 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 482 F1177 GGGGAGGAAGAATT 116 995 R23 AATAACGAATACTT 996 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 483 F1178 GGGAGGAAGAATTG 115 997 R23 AATAACGAATACTT 998 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 484 F1179 GGAGGAAGAATTGG 114 999 R23 AATAACGAATACTT 1000 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 485 F1180 GAGGAAGAATTGGG 113 1001 R23 AATAACGAATACTT 1002 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 486 F1181 AGGAAGAATTGGGT 112 1003 R23 AATAACGAATACTT 1004 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 487 F1182 GGAAGAATTGGGTG 111 1005 R23 AATAACGAATACTT 1006 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 488 F1183 GAAGAATTGGGTGT 110 1007 R23 AATAACGAATACTT 1008 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 489 F1184 AAGAATTGGGTGTG 109 1009 R23 AATAACGAATACTT 1010 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 490 F1185 AGAATTGGGTGTGA 108 1011 R23 AATAACGAATACTT 1012 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 491 F1186 GAATTGGGTGTGAA 107 1013 R23 AATAACGAATACTT 1014 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 492 F1187 AATTGGGTGTGAAA 106 1015 R23 AATAACGAATACTT 1016 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 493 F1188 ATTGGGTGTGAAAT 105 1017 R23 AATAACGAATACTT 1018 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 494 F1189 TTGGGTGTGAAATA 104 1019 R23 AATAACGAATACTT 1020 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 495 F1190 TGGGTGTGAAATAG 103 1021 R23 AATAACGAATACTT 1022 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 496 F1191 GGGTGTGAAATAGT 102 1023 R23 AATAACGAATACTT 1024 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 497 F1192 GGTGTGAAATAGTC 101 1025 R23 AATAACGAATACTT 1026 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 498 F1193 GTGTGAAATAGTCG 100 1027 R23 AATAACGAATACTT 1028 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 499 F1194 TGTGAAATAGTCGA 99 1029 R23 AATAACGAATACTT 1030 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 500 F1195 GTGAAATAGTCGAT 98 1031 R23 AATAACGAATACTT 1032 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 501 F1196 TGAAATAGTCGATT 97 1033 R23 AATAACGAATACTT 1034 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 502 F1197 GAAATAGTCGATTT 96 1035 R23 AATAACGAATACTT 1036 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 503 F1198 AAATAGTCGATTTA 95 1037 R23 AATAACGAATACTT 1038 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 504 F1199 AATAGTCGATTTAT 94 1039 R23 AATAACGAATACTT 1040 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 505 F1200 ATAGTCGATTTATA 93 1041 R23 AATAACGAATACTT 1042 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 506 F1201 TAGTCGATTTATAT 92 1043 R23 AATAACGAATACTT 1044 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 507 F1202 AGTCGATTTATATT 91 1045 R23 AATAACGAATACTT 1046 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 508 F1203 GTCGATTTATATTT 90 1047 R23 AATAACGAATACTT 1048 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 509 F1204 TCGATTTATATTTA 89 1049 R23 AATAACGAATACTT 1050 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 510 F1205 CGATTTATATTTAG 88 1051 R23 AATAACGAATACTT 1052 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 511 F1206 GATTTATATTTAGT 87 1053 R23 AATAACGAATACTT 1054 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 512 F1207 ATTTATATTTAGTA 86 1055 R23 AATAACGAATACTT 1056 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 513 F1208 TTTATATTTAGTAT 85 1057 R23 AATAACGAATACTT 1058 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 514 F1209 TTATATTTAGTATT 84 1059 R23 AATAACGAATACTT 1060 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 515 F1210 TATATTTAGTATTA 83 1061 R23 AATAACGAATACTT 1062 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 516 F1211 ATATTTAGTATTAG 82 1063 R23 AATAACGAATACTT 1064 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 517 F1212 TATTTAGTATTAGG 81 1065 R23 AATAACGAATACTT 1066 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 518 F1213 ATTTAGTATTAGGA 80 1067 R23 AATAACGAATACTT 1068 P23 GAGCGAGGGAGAAGTTGGGGAGGAGA 968 519 F1214 TTTAGTATTAGGAC 140 1069 R24 CGCAATCGACAACC 1070 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 520 F1215 TTAGTATTAGGACG 139 1072 R24 CGCAATCGACAACC 1073 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 521 F1216 TAGTATTAGGACGT 138 1074 R24 CGCAATCGACAACC 1075 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 522 F1217 AGTATTAGGACGTA 137 1076 R24 CGCAATCGACAACC 1077 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 523 F1218 GTATTAGGACGTAG 136 1078 R24 CGCAATCGACAACC 1079 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 524 F1219 TATTAGGACGTAGG 135 1080 R24 CGCAATCGACAACC 1081 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 525 F1220 ATTAGGACGTAGGG 134 1082 R24 CGCAATCGACAACC 1083 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 526 F1221 TTAGGACGTAGGGT 133 1084 R24 CGCAATCGACAACC 1085 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 527 F1222 TAGGACGTAGGGTT 132 1086 R24 CGCAATCGACAACC 1087 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 528 F1223 AGGACGTAGGGTTT 131 1088 R24 CGCAATCGACAACC 1089 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 529 F1224 GGACGTAGGGTTTT 130 1090 R24 CGCAATCGACAACC 1091 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 530 F1225 GACGTAGGGTTTTA 129 1092 R24 CGCAATCGACAACC 1093 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 531 F1226 ACGTAGGGTTTTAC 128 1094 R24 CGCAATCGACAACC 1095 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 532 F1227 CGTAGGGTTTTACG 127 1096 R24 CGCAATCGACAACC 1097 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 533 F1228 GTAGGGTTTTACGA 126 1098 R24 CGCAATCGACAACC 1099 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 534 F1229 TAGGGTTTTACGAG 125 1100 R24 CGCAATCGACAACC 1101 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 535 F1230 AGGGTTTTACGAGT 124 1102 R24 CGCAATCGACAACC 1103 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 536 F1231 GGGTTTTACGAGTT 123 1104 R24 CGCAATCGACAACC 1105 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 537 F1232 GGTTTTACGAGTTA 122 1106 R24 CGCAATCGACAACC 1107 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 538 F1233 GTTTTACGAGTTAT 121 1108 R24 CGCAATCGACAACC 1109 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 539 F1234 TTTTACGAGTTATT 120 1110 R24 CGCAATCGACAACC 1111 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 540 F1235 TTTACGAGTTATTT 119 1112 R24 CGCAATCGACAACC 1113 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 541 F1236 TTACGAGTTATTTC 118 1114 R24 CGCAATCGACAACC 1115 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 542 F1237 TACGAGTTATTTCG 117 1116 R24 CGCAATCGACAACC 1117 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 543 F1238 ACGAGTTATTTCGA 116 1118 R24 CGCAATCGACAACC 1119 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 544 F1239 CGAGTTATTTCGAA 115 1120 R24 CGCAATCGACAACC 1121 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 545 F1240 GAGTTATTTCGAAG 114 1122 R24 CGCAATCGACAACC 1123 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 546 F1241 AGTTATTTCGAAGA 113 1124 R24 CGCAATCGACAACC 1125 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 547 F1242 GTTATTTCGAAGAG 112 1126 R24 CGCAATCGACAACC 1127 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 548 F1243 TTATTTCGAAGAGC 111 1128 R24 CGCAATCGACAACC 1129 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 549 F1244 TATTTCGAAGAGCG 110 1130 R24 CGCAATCGACAACC 1131 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 550 F1245 ATTTCGAAGAGCGA 109 1132 R24 CGCAATCGACAACC 1133 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 551 F1246 TTTCGAAGAGCGAG 108 1134 R24 CGCAATCGACAACC 1135 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 552 F1247 TTCGAAGAGCGAGG 107 1136 R24 CGCAATCGACAACC 1137 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 553 F1248 TCGAAGAGCGAGGG 106 1138 R24 CGCAATCGACAACC 1139 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 554 F1249 CGAAGAGCGAGGGA 105 1140 R24 CGCAATCGACAACC 1141 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 555 F1250 GAAGAGCGAGGGAG 104 1142 R24 CGCAATCGACAACC 1143 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 556 F1251 AAGAGCGAGGGAGA 103 1144 R24 CGCAATCGACAACC 1145 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 557 F1252 AGAGCGAGGGAGAA 102 1146 R24 CGCAATCGACAACC 1147 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 558 F1253 GAGCGAGGGAGAAG 101 1148 R24 CGCAATCGACAACC 1149 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 559 F1254 AGCGAGGGAGAAGT 100 1150 R24 CGCAATCGACAACC 1151 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 560 F1255 GCGAGGGAGAAGTT 99 1152 R24 CGCAATCGACAACC 1153 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 561 F1256 CGAGGGAGAAGTTG 98 1154 R24 CGCAATCGACAACC 1155 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 562 F1257 GAGGGAGAAGTTGG 97 1156 R24 CGCAATCGACAACC 1157 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 563 F1258 AGGGAGAAGTTGGG 96 1158 R24 CGCAATCGACAACC 1159 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 564 F1259 GGGAGAAGTTGGGG 95 1160 R24 CGCAATCGACAACC 1161 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 565 F1260 GGAGAAGTTGGGGA 94 1162 R24 CGCAATCGACAACC 1163 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 566 F1261 GAGAAGTTGGGGAG 93 1164 R24 CGCAATCGACAACC 1165 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 567 F1262 AGAAGTTGGGGAGG 92 1166 R24 CGCAATCGACAACC 1167 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 568 F1263 GAAGTTGGGGAGGA 91 1168 R24 CGCAATCGACAACC 1169 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 569 F1264 AAGTTGGGGAGGAG 90 1170 R24 CGCAATCGACAACC 1171 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 570 F1265 AGTTGGGGAGGAGA 89 1172 R24 CGCAATCGACAACC 1173 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 571 F1266 GTTGGGGAGGAGAA 88 1174 R24 CGCAATCGACAACC 1175 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 572 F1267 TTGGGGAGGAGAAA 87 1176 R24 CGCAATCGACAACC 1177 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 573 F1268 TGGGGAGGAGAAAG 86 1178 R24 CGCAATCGACAACC 1179 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 574 F1269 GGGGAGGAGAAAGT 85 1180 R24 CGCAATCGACAACC 1181 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 575 F1270 GGGAGGAGAAAGTA 84 1182 R24 CGCAATCGACAACC 1183 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 576 F1271 GGAGGAGAAAGTAT 83 1184 R24 CGCAATCGACAACC 1185 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 577 F1272 GAGGAGAAAGTATT 82 1186 R24 CGCAATCGACAACC 1187 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 578 F1273 AGGAGAAAGTATTC 81 1188 R24 CGCAATCGACAACC 1189 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 579 F1274 GGAGAAAGTATTCG 80 1190 R24 CGCAATCGACAACC 1191 P24 GTAGGGGAGATGTTGTTTTTTCGC 1071 580 F1275 GAGAAAGTATTCGT 120 1192 R25 ACGAACTAAAATTA 1193 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 581 F1276 AGAAAGTATTCGTT 119 1195 R25 ACGAACTAAAATTA 1196 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 582 F1277 GAAAGTATTCGTTA 118 1197 R25 ACGAACTAAAATTA 1198 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 583 F1278 AAAGTATTCGTTAT 117 1199 R25 ACGAACTAAAATTA 1200 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 584 F1279 AAGTATTCGTTATT 116 1201 R25 ACGAACTAAAATTA 1202 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 585 F1280 AGTATTCGTTATTT 115 1203 R25 ACGAACTAAAATTA 1204 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 586 F1281 GTATTCGTTATTTT 114 1205 R25 ACGAACTAAAATTA 1206 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 587 F1282 TATTCGTTATTTTT 113 1207 R25 ACGAACTAAAATTA 1208 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 588 F1283 ATTCGTTATTTTTG 112 1209 R25 ACGAACTAAAATTA 1210 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 589 F1284 TTCGTTATTTTTGG 111 1211 R25 ACGAACTAAAATTA 1212 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 590 F1285 TCGTTATTTTTGGA 110 1213 R25 ACGAACTAAAATTA 1214 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 591 F1286 CGTTATTTTTGGAT 109 1215 R25 ACGAACTAAAATTA 1216 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 592 F1287 GTTATTTTTGGATT 108 1217 R25 ACGAACTAAAATTA 1218 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 593 F1288 TTATTTTTGGATTG 107 1219 R25 ACGAACTAAAATTA 1220 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 594 F1289 TATTTTTGGATTGG 106 1221 R25 ACGAACTAAAATTA 1222 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 595 F1290 ATTTTTGGATTGGC 105 1223 R25 ACGAACTAAAATTA 1224 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 596 F1291 TTTTTGGATTGGCG 104 1225 R25 ACGAACTAAAATTA 1226 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 597 F1292 TTTTGGATTGGCGT 103 1227 R25 ACGAACTAAAATTA 1228 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 598 F1293 TTTGGATTGGCGTA 102 1229 R25 ACGAACTAAAATTA 1230 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 599 F1294 TTGGATTGGCGTAT 101 1231 R25 ACGAACTAAAATTA 1232 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 600 F1295 TGGATTGGCGTATT 100 1233 R25 ACGAACTAAAATTA 1234 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 601 F1296 GGATTGGCGTATTT 99 1235 R25 ACGAACTAAAATTA 1236 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 602 F1297 GATTGGCGTATTTA 98 1237 R25 ACGAACTAAAATTA 1238 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 603 F1298 ATTGGCGTATTTAT 97 1239 R25 ACGAACTAAAATTA 1240 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 604 F1299 TTGGCGTATTTATA 96 1241 R25 ACGAACTAAAATTA 1242 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 605 F1300 TGGCGTATTTATAG 95 1243 R25 ACGAACTAAAATTA 1244 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 606 F1301 GGCGTATTTATAGG 94 1245 R25 ACGAACTAAAATTA 1246 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 607 F1302 GCGTATTTATAGGC 93 1247 R25 ACGAACTAAAATTA 1248 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 608 F1303 CGTATTTATAGGCG 92 1249 R25 ACGAACTAAAATTA 1250 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 609 F1304 GTATTTATAGGCGT 91 1251 R25 ACGAACTAAAATTA 1252 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 610 F1305 TATTTATAGGCGTA 90 1253 R25 ACGAACTAAAATTA 1254 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 611 F1306 ATTTATAGGCGTAG 89 1255 R25 ACGAACTAAAATTA 1256 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 612 F1307 TTTATAGGCGTAGG 88 1257 R25 ACGAACTAAAATTA 1258 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 613 F1308 TTATAGGCGTAGGG 87 1259 R25 ACGAACTAAAATTA 1260 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 614 F1309 TATAGGCGTAGGGG 86 1261 R25 ACGAACTAAAATTA 1262 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 615 F1310 ATAGGCGTAGGGGA 85 1263 R25 ACGAACTAAAATTA 1264 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 616 F1311 TAGGCGTAGGGGAG 84 1265 R25 ACGAACTAAAATTA 1266 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 617 F1312 AGGCGTAGGGGAGA 83 1267 R25 ACGAACTAAAATTA 1268 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 618 F1313 GGCGTAGGGGAGAT 82 1269 R25 ACGAACTAAAATTA 1270 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 619 F1314 GCGTAGGGGAGATG 81 1271 R25 ACGAACTAAAATTA 1272 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 620 F1315 CGTAGGGGAGATGT 80 1273 R25 ACGAACTAAAATTA 1274 P25 AGTTCGTAGTTCGAGTTATTTTTT 1194 621 F1316 GTAGGGGAGATGTT 120 1275 R26 AAAAAAAAAAAACG 1276 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 622 F1317 TAGGGGAGATGTTG 119 1278 R26 AAAAAAAAAAAACG 1279 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 623 F1318 AGGGGAGATGTTGT 118 1280 R26 AAAAAAAAAAAACG 1281 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 624 F1319 GGGGAGATGTTGTT 117 1282 R26 AAAAAAAAAAAACG 1283 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 625 F1320 GGGAGATGTTGTTT 116 1284 R26 AAAAAAAAAAAACG 1285 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 626 F1321 GGAGATGTTGTTTT 115 1286 R26 AAAAAAAAAAAACG 1287 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 627 F1322 GAGATGTTGTTTTT 114 1288 R26 AAAAAAAAAAAACG 1289 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 628 F1323 AGATGTTGTTTTTT 113 1290 R26 AAAAAAAAAAAACG 1291 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 629 F1324 GATGTTGTTTTTTC 112 1292 R26 AAAAAAAAAAAACG 1293 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 630 F1325 ATGTTGTTTTTTCG 111 1294 R26 AAAAAAAAAAAACG 1295 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 631 F1326 TGTTGTTTTTTCGC 110 1296 R26 AAAAAAAAAAAACG 1297 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 632 F1327 GTTGTTTTTTCGCG 109 1298 R26 AAAAAAAAAAAACG 1299 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 633 F1328 TTGTTTTTTCGCGG 108 1300 R26 AAAAAAAAAAAACG 1301 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 634 F1329 TGTTTTTTCGCGGT 107 1302 R26 AAAAAAAAAAAACG 1303 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 635 F1330 GTTTTTTCGCGGTT 106 1304 R26 AAAAAAAAAAAACG 1305 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 636 F1331 TTTTTTCGCGGTTG 105 1306 R26 AAAAAAAAAAAACG 1307 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 637 F1332 TTTTTCGCGGTTGT 104 1308 R26 AAAAAAAAAAAACG 1309 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 638 F1333 TTTTCGCGGTTGTC 103 1310 R26 AAAAAAAAAAAACG 1311 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 639 F1334 TTTCGCGGTTGTCG 102 1312 R26 AAAAAAAAAAAACG 1313 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 640 F1335 TTCGCGGTTGTCGA 101 1314 R26 AAAAAAAAAAAACG 1315 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 641 F1336 TCGCGGTTGTCGAT 100 1316 R26 AAAAAAAAAAAACG 1317 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 642 F1337 CGCGGTTGTCGATT 99 1318 R26 AAAAAAAAAAAACG 1319 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 643 F1338 GCGGTTGTCGATTG 98 1320 R26 AAAAAAAAAAAACG 1321 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 644 F1339 CGGTTGTCGATTGC 97 1322 R26 AAAAAAAAAAAACG 1323 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 645 F1340 GGTTGTCGATTGCG 96 1324 R26 AAAAAAAAAAAACG 1325 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 646 F1341 GTTGTCGATTGCGT 95 1326 R26 AAAAAAAAAAAACG 1327 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 647 F1342 TTGTCGATTGCGTT 94 1328 R26 AAAAAAAAAAAACG 1329 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 648 F1343 TGTCGATTGCGTTT 93 1330 R26 AAAAAAAAAAAACG 1331 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 649 F1344 GTCGATTGCGTTTA 92 1332 R26 AAAAAAAAAAAACG 1333 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 650 F1345 TCGATTGCGTTTAG 91 1334 R26 AAAAAAAAAAAACG 1335 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 651 F1346 CGATTGCGTTTAGT 90 1336 R26 AAAAAAAAAAAACG 1337 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 652 F1347 GATTGCGTTTAGTT 89 1338 R26 AAAAAAAAAAAACG 1339 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 653 F1348 ATTGCGTTTAGTTC 88 1340 R26 AAAAAAAAAAAACG 1341 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 654 F1349 TTGCGTTTAGTTCG 87 1342 R26 AAAAAAAAAAAACG 1343 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 655 F1350 TGCGTTTAGTTCGT 86 1344 R26 AAAAAAAAAAAACG 1345 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 656 F1351 GCGTTTAGTTCGTA 85 1346 R26 AAAAAAAAAAAACG 1347 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 657 F1352 CGTTTAGTTCGTAG 84 1348 R26 AAAAAAAAAAAACG 1349 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 658 F1353 GTTTAGTTCGTAGT 83 1350 R26 AAAAAAAAAAAACG 1351 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 659 F1354 TTTAGTTCGTAGTT 82 1352 R26 AAAAAAAAAAAACG 1353 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 660 F1355 TTAGTTCGTAGTTC 81 1354 R26 AAAAAAAAAAAACG 1355 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 661 F1356 TAGTTCGTAGTTCG 80 1356 R26 AAAAAAAAAAAACG 1357 P26 ATTTTTTTTAGTTAAGTTTATTGGG 1277 662 F1357 AGTTCGTAGTTCGA 150 1358 R27 CGAACACGTCAATC 1359 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 663 F1358 GTTCGTAGTTCGAG 149 1361 R27 CGAACACGTCAATC 1362 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 664 F1359 TTCGTAGTTCGAGT 148 1363 R27 CGAACACGTCAATC 1364 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 665 F1360 TCGTAGTTCGAGTT 147 1365 R27 CGAACACGTCAATC 1366 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 666 F1361 CGTAGTTCGAGTTA 146 1367 R27 CGAACACGTCAATC 1368 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 667 F1362 GTAGTTCGAGTTAT 145 1369 R27 CGAACACGTCAATC 1370 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 668 F1363 TAGTTCGAGTTATT 144 1371 R27 CGAACACGTCAATC 1372 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 669 F1364 AGTTCGAGTTATTT 143 1373 R27 CGAACACGTCAATC 1374 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 670 F1365 GTTCGAGTTATTTT 142 1375 R27 CGAACACGTCAATC 1376 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 671 F1366 TTCGAGTTATTTTT 141 1377 R27 CGAACACGTCAATC 1378 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 672 F1367 TCGAGTTATTTTTT 140 1379 R27 CGAACACGTCAATC 1380 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 673 F1368 CGAGTTATTTTTTT 139 1381 R27 CGAACACGTCAATC 1382 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 674 F1369 GAGTTATTTTTTTA 138 1383 R27 CGAACACGTCAATC 1384 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 675 F1370 AGTTATTTTTTTAA 137 1385 R27 CGAACACGTCAATC 1386 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 676 F1371 GTTATTTTTTTAAT 136 1387 R27 CGAACACGTCAATC 1388 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 677 F1372 TTATTTTTTTAATT 135 1389 R27 CGAACACGTCAATC 1390 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 678 F1373 TATTTTTTTAATTT 134 1391 R27 CGAACACGTCAATC 1392 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 679 F1374 ATTTTTTTAATTTT 133 1393 R27 CGAACACGTCAATC 1394 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 680 F1375 TTTTTTTAATTTTA 132 1395 R27 CGAACACGTCAATC 1396 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 681 F1376 TTTTTTAATTTTAG 131 1397 R27 CGAACACGTCAATC 1398 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 682 F1377 TTTTTAATTTTAGT 130 1399 R27 CGAACACGTCAATC 1400 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 683 F1378 TTTTAATTTTAGTT 129 1401 R27 CGAACACGTCAATC 1402 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 684 F1379 TTTAATTTTAGTTC 128 1403 R27 CGAACACGTCAATC 1404 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 685 F1380 TTAATTTTAGTTCG 127 1405 R27 CGAACACGTCAATC 1406 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360 686 F1381 TAATTTTAGTTCGT 126 1407 R27 CGAACACGTCAATC 1408 P27 TTTAAGAGTAAAGGAGGTTTTGGG 1360

As a result of evaluating methylation of GPM6A gene using DNA from colorectal cancer tissues and normal tissues adjacent to cancer tissues, it was found that the sensitivity of GPM6A gene for colorectal cancer diagnosis was 70% (14/20)˜90.0% (18/20) and the specificity of the GPM6A gene was 80% (4/20)˜95% (1/20). Such results suggest that the GPM6A methylation biomarker gene is useful for diagnosis of colorectal cancer.

TABLE 8 Evaluation of ability to diagnose colorectal cancer using GPM6A gene Set of primers Sensitivity (%), Specificity (%), and probes Cut-off (Ct) n = 20 n = 20 1 >3.3 90 85 2 >2.4 85 90 3 >5.7 90 85 4 >3.3 85 90 5 >1.2 85 85 6 >0.4 80 90 7 >2.1 85 85 8 >5.7 80 90 9 >2.3 75 90 10 >0.5 75 80 11 >1.2 80 85 12 >2.1 90 85 13 >3.0 80 90 14 >3.2 85 90 15 >0.6 85 85 16 >1.2 85 85 17 >5.7 90 80 18 >2.9 80 95 19 >0.4 75 90 20 >3.5 80 85 21 >0.7 90 85 22 >4.2 85 85 23 >3.0 80 90 24 >0.4 90 90 25 >2.9 80 90 26 >3.6 85 90 27 >1.2 85 90 28 >2.2 80 95 29 >3.8 75 95 30 >2.9 80 90 31 >0.7 85 85 32 >3.6 80 95 33 >2.2 80 90 34 >3.3 75 90 35 >2.1 90 85 36 >0.4 90 80 37 >2.2 75 95 38 >0.4 85 90 39 >2.2 80 90 40 >2.7 75 95 41 >1.2 75 90 42 >0.4 85 85 43 >0.4 90 85 44 >0.7 90 80 45 >1.2 80 90 46 >2.0 80 90 47 >2.1 80 90 48 >2.1 80 85 49 >2.2 80 90 50 >2.3 90 80 51 >2.3 75 95 52 >2.7 85 90 53 >2.7 80 90 54 >3.2 85 85 55 >3.3 85 90 56 >3.6 85 85 57 >4.2 85 90 58 >4.5 80 85 59 >5.7 80 95 60 >2.1 90 85 61 >2.1 85 90 62 >0.4 85 90 63 >5.7 85 90 64 >2.3 85 90 65 >4.3 90 90 66 >2.1 85 90 67 >0.7 80 90 68 >0.5 75 90 69 >3.3 75 95 70 >0.4 75 95 71 >0.5 85 90 72 >0.7 85 90 73 >1.2 75 90 74 >2.0 75 90 75 >2.1 75 85 76 >2.2 85 90 77 >2.2 80 90 78 >2.3 80 90 79 >2.3 85 90 80 >2.7 85 90 81 >2.9 80 90 82 >3.2 90 80 83 >3.3 85 90 84 >3.6 75 95 85 >4.2 80 85 86 >4.5 75 90 87 >5.7 80 90 88 >0.5 85 90 89 >1.2 80 85 90 >2.4 90 85 91 >2.1 80 95 92 >4.2 80 85 93 >0.5 80 95 94 >0.7 85 90 95 >3.5 80 90 96 >2.4 80 90 97 >2.7 85 85 98 >2.4 75 90 99 >2.1 80 90 100 >4.2 90 80 101 >4.5 80 85 102 >4.2 85 90 103 >3.6 85 90 104 >0.7 90 85 105 >2.1 85 90 106 >2.7 85 90 107 >3.2 80 95 108 >2.9 85 90 109 >2.2 75 90 110 >1.2 85 90 111 >0.4 85 90 112 >2.7 80 85 113 >0.4 90 85 114 >2.3 75 95 115 >2.3 85 85 116 >1.2 75 95 117 >2.1 90 80 118 >0.4 80 95 119 >2.3 75 90 120 >2.3 80 85 121 >2.3 85 90 122 >2.3 80 85 123 >0.5 80 85 124 >2.3 85 85 125 >2.2 85 85 126 >2.7 75 90 127 >2.3 85 95 128 >3.2 80 90 129 >2.1 85 85 130 >5.7 80 90 131 >3.3 85 90 132 >2.4 75 90 133 >0.4 80 85 134 >0.5 85 90 135 >2.3 85 90 136 >2.3 75 90 137 >5.7 80 95 138 >2.3 90 85 139 >2.0 80 85 140 >3.3 85 95 141 >0.4 85 90 142 >0.4 80 90 143 >0.4 80 85 144 >0.5 85 90 145 >0.7 85 90 146 >1.2 80 95 147 >1.2 80 90 148 >2.0 80 95 149 >2.1 75 90 150 >4.1 90 85 151 >2.1 80 90 152 >2.2 85 85 153 >2.3 80 90 154 >3.2 80 90 155 >4.7 80 90 156 >2.3 80 85 157 >2.4 90 85 158 >2.7 90 85 159 >2.7 80 95 160 >2.9 80 90 161 >3.0 85 85 162 >3.2 85 90 163 >3.5 90 85 164 >3.5 80 90 165 >3.6 75 90 166 >4.2 80 95 167 >4.3 85 90 168 >4.5 75 90 169 >5.7 80 90 170 >2.2 75 90 171 >3.6 80 90 172 >2.9 85 85 173 >4.3 80 90 174 >3.0 90 80 175 >4.3 75 90 176 >3.3 85 90 177 >2.0 90 85 178 >2.3 85 90 179 >2.4 75 95 180 >3.3 90 85 181 >2.3 85 90 182 >2.3 75 95 183 >0.7 75 90 184 >2.1 80 90 185 >4.5 75 90 186 >0.4 80 90 187 >0.4 85 90 188 >0.4 80 90 189 >0.5 80 85 190 >0.7 80 85 191 >1.2 85 90 192 >1.2 90 85 193 >2.3 85 90 194 >2.1 80 90 195 >5.1 85 90 196 >2.1 90 85 197 >2.1 85 90 198 >2.2 90 85 199 >2.3 85 85 200 >3.6 85 90 201 >2.3 85 90 202 >2.4 80 90 203 >2.7 75 90 204 >2.7 80 85 205 >2.9 80 85 206 >3.0 85 85 207 >3.2 80 90 208 >3.3 90 85 209 >3.5 80 90 210 >3.6 90 85 211 >4.2 85 90 212 >4.3 75 90 213 >4.5 85 90 214 >5.7 85 95 215 >2.1 75 90 216 >2.3 75 95 217 >1.2 80 90 218 >2.1 80 90 219 >3.0 85 85 220 >4.3 75 90 221 >2.7 80 90 222 >2.3 85 90 223 >2.3 75 90 224 >2.9 85 90 225 >3.0 85 90 226 >2.9 85 90 227 >0.5 80 90 228 >4.3 75 95 229 >2.7 90 90 230 >1.2 85 90 231 >5.7 85 90 232 >2.3 75 95 233 >0.4 85 85 234 >2.3 85 90 235 >4.2 85 90 236 >1.2 80 90 237 >4.2 85 90 238 >2.2 80 90 239 >0.5 90 85 240 >3.0 75 90 241 >2.9 85 90 242 >2.3 75 90 243 >2.3 85 85 244 >2.7 85 90 245 >3.3 80 85 246 >2.0 90 85 247 >4.2 85 90 248 >3.5 90 85 249 >3.2 80 90 250 >0.4 75 90 251 >3.6 85 90 252 >0.5 85 85 253 >4.5 90 85 254 >2.3 85 90 255 >2.0 75 95 256 >3.5 90 85 257 >0.5 80 90 258 >2.7 85 90 259 >3.0 85 85 260 >2.9 80 90 261 >3.2 85 85 262 >3.5 75 95 263 >4.3 85 85 264 >2.1 75 95 265 >1.2 80 95 266 >4.3 80 90 267 >4.3 85 90 268 >2.1 80 90 269 >2.3 75 95 270 >4.5 80 90 271 >3.0 85 90 272 >2.1 80 90 273 >0.4 80 85 274 >0.4 80 90 275 >2.1 90 80 276 >0.6 80 90 277 >0.5 85 85 278 >0.5 90 85 279 >1.2 80 90 280 >1.2 85 90 281 >2.0 90 80 282 >2.1 80 95 283 >2.1 80 90 284 >2.1 80 90 285 >2.3 85 90 286 >2.3 80 95 287 >2.4 75 90 288 >2.4 85 85 289 >2.8 75 90 290 >2.9 85 90 291 >3.2 75 95 292 >3.3 85 85 293 >3.6 90 80 294 >3.8 75 90 295 >4.3 85 90 296 >4.5 90 85 297 >2.1 80 90 298 >1.2 80 95 299 >4.5 90 85 300 >2.4 85 90 301 >2.1 80 90 302 >2.1 75 90 303 >5.7 80 90 304 >4.5 90 80 305 >2.7 85 90 306 >4.3 85 85 307 >0.4 85 90 308 >2.1 75 90 309 >4.5 75 85 310 >0.4 75 95 311 >0.5 75 85 312 >0.5 80 90 313 >1.2 75 90 314 >1.2 80 90 315 >2.0 80 90 316 >2.1 85 95 317 >2.1 80 90 318 >2.2 85 90 319 >2.3 75 90 320 >5.2 85 85 321 >2.4 75 85 322 >2.4 90 85 323 >2.7 80 90 324 >2.9 85 90 325 >3.2 85 90 326 >3.3 85 85 327 >3.6 80 90 328 >3.8 75 80 329 >4.3 85 85 330 >4.5 80 90 331 >0.7 90 85 332 >3.3 90 85 333 >4.5 85 90 334 >2.3 80 90 335 >1.2 85 90 336 >2.4 90 80 337 >2.1 85 85 338 >2.7 85 90 339 >1.2 85 90 340 >1.2 85 95 341 >2.7 85 85 342 >2.3 80 85 343 >3.5 90 85 344 >2.1 85 90 345 >3.3 75 85 346 >4.3 90 85 347 >0.5 75 90 348 >2.3 80 85 349 >2.0 75 90 350 >0.4 80 85 351 >0.7 80 90 352 >0.7 85 85 353 >3.2 90 80 354 >3.6 85 85 355 >2.3 85 85 356 >0.4 75 90 357 >3.6 75 90 358 >4.3 75 95 359 >2.4 75 95 360 >4.5 80 90 361 >2.9 75 95 362 >5.6 75 90 363 >2.3 75 95 364 >0.5 85 85 365 >2.1 85 90 366 >4.2 85 85 367 >2.7 90 85 368 >2.1 75 90 369 >2.0 85 85 370 >5.7 90 90 371 >4.5 80 90 372 >1.2 80 85 373 >4.2 85 85 374 >3.3 85 90 375 >1.2 90 85 376 >0.7 75 90 377 >2.3 75 95 378 >3.6 90 85 379 >0.4 75 90 380 >0.5 85 90 381 >0.7 80 90 382 >1.2 85 95 383 >2.0 85 90 384 >2.1 85 90 385 >2.2 80 90 386 >2.3 85 90 387 >2.3 85 90 388 >2.7 85 85 389 >2.9 80 90 390 >3.2 85 95 391 >3.5 90 85 392 >4.2 90 80 393 >4.3 80 90 394 >5.7 85 90 395 >2.1 75 95 396 >2.3 80 85 397 >0.4 75 90 398 >1.2 80 85 399 >2.4 75 90 400 >2.7 85 90 401 >0.4 80 90 402 >0.4 80 90 403 >1.2 85 85 404 >0.4 90 85 405 >0.4 85 85 406 >0.7 85 85 407 >1.2 75 95 408 >2.0 85 95 409 >2.1 80 90 410 >2.2 85 85 411 >2.3 85 90 412 >2.3 75 80 413 >2.4 90 85 414 >2.7 90 85 415 >3.0 75 90 416 >3.5 80 90 417 >3.6 90 85 418 >4.3 75 90 419 >4.5 85 90 420 >0.4 90 80 421 >2.1 80 85 422 >5.7 85 85 423 >2.3 75 95 424 >2.4 75 95 425 >5.7 85 85 426 >2.1 75 90 427 >0.5 80 90 428 >2.1 90 85 429 >3.6 85 90 430 >4.3 75 95 431 >1.2 75 90 432 >2.3 75 90 433 >0.4 85 90 434 >2.7 90 80 435 >2.2 85 85 436 >2.7 75 95 437 >4.5 75 90 438 >2.7 80 90 439 >3.5 75 95 440 >5.4 80 90 441 >2.3 85 85 442 >3.2 90 85 443 >2.3 75 90 444 >0.4 85 90 445 >2.9 80 85 446 >1.2 90 85 447 >3.6 90 80 448 >2.3 85 90 449 >2.1 85 85 450 >3.0 80 95 451 >5.7 90 80 452 >2.1 85 85 453 >2.7 90 85 454 >0.4 75 95 455 >0.7 85 90 456 >2.3 85 85 457 >2.1 75 95 458 >2.3 75 95 459 >2.4 85 85 460 >3.0 85 85 461 >3.3 80 85 462 >4.2 90 80 463 >5.7 75 95 464 >5.7 75 95 465 >2.3 80 90 466 >4.2 85 90 467 >3.0 75 95 468 >0.4 90 80 469 >0.5 80 85 470 >0.5 75 90 471 >1.2 85 90 472 >2.1 85 85 473 >2.3 90 80 474 >2.3 90 85 475 >2.7 85 90 476 >3.2 75 90 477 >3.6 85 90 478 >5.6 75 90 479 >2.0 85 90 480 >2.1 75 95 481 >3.3 85 90 482 >2.1 75 90 483 >0.5 85 95 484 >2.7 85 90 485 >3.0 80 90 486 >3.0 75 90 487 >3.3 80 95 488 >0.4 80 90 489 >4.5 90 85 490 >4.2 75 95 491 >2.2 80 95 492 >2.2 80 85 493 >3.3 85 90 494 >2.4 75 90 495 >4.5 80 90 496 >0.5 75 90 497 >3.6 75 90 498 >0.4 85 85 499 >2.0 75 95 500 >0.5 85 90 501 >3.3 75 90 502 >4.5 80 90 503 >0.5 85 90 504 >2.1 75 90 505 >2.2 75 95 506 >2.7 75 95 507 >3.0 80 90 508 >4.3 85 90 509 >4.3 85 90 510 >4.2 90 85 511 >2.2 90 85 512 >2.1 80 95 513 >0.4 90 80 514 >2.1 85 85 515 >2.1 75 95 516 >2.7 75 90 517 >3.0 85 90 518 >4.3 90 85 519 >5.7 80 90 520 >2.7 80 90 521 >3.3 85 90 522 >1.2 80 90 523 >0.5 75 90 524 >4.3 80 85 525 >2.1 90 85 526 >0.5 80 85 527 >2.3 80 90 528 >3.6 80 90 529 >2.3 80 90 530 >2.4 80 95 531 >0.4 85 90 532 >2.3 85 90 533 >0.4 90 85 534 >1.2 85 85 535 >2.1 75 90 536 >2.4 80 90 537 >2.7 85 90 538 >4.2 80 85 539 >5.7 85 85 540 >3.0 75 95 541 >3.3 85 85 542 >2.7 85 85 543 >0.5 85 90 544 >2.1 90 90 545 >2.3 90 80 546 >3.0 85 90 547 >3.5 75 90 548 >4.2 80 95 549 >2.9 75 90 550 >5.7 80 90 551 >2.1 85 85 552 >3.0 80 90 553 >2.3 80 90 554 >0.4 85 90 555 >2.7 75 95 556 >2.2 90 80 557 >3.6 80 90 558 >2.3 85 85 559 >3.5 80 90 560 >3.2 85 90 561 >4.5 85 90 562 >0.4 85 90 563 >2.2 85 90 564 >2.3 80 85 565 >3.5 80 95 566 >4.3 85 85 567 >1.2 85 85 568 >4.2 85 85 569 >0.4 90 80 570 >1.2 80 95 571 >2.3 85 90 572 >2.7 80 90 573 >4.2 90 80 574 >5.7 80 95 575 >4.5 80 90 576 >2.3 90 90 577 >2.1 80 90 578 >2.4 85 90 579 >0.4 85 90 580 >3.6 85 85 581 >0.5 70 85 582 >2.1 85 90 583 >4.3 85 90 584 >2.4 85 90 585 >0.4 80 90 586 >1.2 75 95 587 >2.3 75 90 588 >3.0 85 95 589 >4.5 85 90 590 >1.2 75 90 591 >2.1 90 85 592 >0.4 75 90 593 >2.0 80 90 594 >2.3 75 90 595 >3.3 80 90 596 >4.3 80 95 597 >0.4 75 95 598 >3.6 85 85 599 >4.2 85 90 600 >2.1 80 90 601 >2.1 85 90 602 >2.7 80 90 603 >3.6 80 90 604 >4.5 85 90 605 >1.2 80 90 606 >3.0 80 90 607 >3.3 80 90 608 >0.5 80 90 609 >2.7 75 90 610 >4.3 85 85 611 >2.1 80 90 612 >2.3 75 95 613 >2.7 90 85 614 >2.7 85 90 615 >0.7 80 90 616 >0.5 80 90 617 >2.1 80 90 618 >2.4 85 90 619 >3.6 85 90 620 >2.7 75 95 621 >0.4 80 85 622 >0.7 80 90 623 >1.2 75 90 624 >2.1 80 90 625 >2.9 80 90 626 >3.6 75 90 627 >4.2 85 85 628 >4.3 80 85 629 >4.3 85 95 630 >5.7 90 85 631 >0.7 90 85 632 >2.3 85 90 633 >5.6 85 90 634 >2.2 80 90 635 >2.3 80 90 636 >3.6 85 90 637 >0.4 75 85 638 >2.3 80 90 639 >2.7 90 85 640 >2.1 85 90 641 >2.2 75 90 642 >2.4 90 85 643 >2.0 85 90 644 >5.7 90 85 645 >0.7 85 90 646 >3.5 80 90 647 >0.5 80 90 648 >2.3 85 90 649 >5.7 85 90 650 >2.7 85 90 651 >2.0 90 85 652 >2.4 85 90 653 >2.4 85 90 654 >2.7 85 90 655 >2.1 90 80 656 >3.3 75 90 657 >2.7 90 85 658 >0.6 80 85 659 >5.7 90 80 660 >3.2 80 85 661 >2.2 80 90 662 >2.3 80 90 663 >2.3 85 85 664 >3.5 90 80 665 >2.1 85 90 666 >2.9 75 90 667 >2.1 75 95 668 >2.1 90 85 669 >3.5 80 90 670 >5.7 75 95 671 >1.2 85 85 672 >4.2 75 95 673 >2.3 75 90 674 >4.5 80 90 675 >2.3 80 90 676 >3.5 80 95 677 >0.4 75 90 678 >2.2 75 95 679 >5.7 85 90 680 >3.3 85 90 681 >0.5 85 90 682 >2.3 80 90 683 >2.2 85 90 684 >0.7 85 90 685 >3.2 80 90 686 >0.7 80 90

INDUSTRIAL APPLICABILITY

As described above, the present disclosure enables the methylation of the CpG island of a colorectal cancer-specific marker gene to be detected to thereby provide information for diagnosing colorectal cancer. The use of the inventive method for detecting methylation and the inventive composition, kit and nucleic acid chip for diagnosing colorectal cancer makes it possible to diagnose colorectal cancer at an early transformation stage, thus enabling the early diagnosis of colorectal cancer. In addition, the inventive method enables colorectal cancer to be effectively diagnosed in an accurate and rapid manner compared to conventional methods.

Although the present disclosure has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present disclosure. Thus, the substantial scope of the present disclosure will be defined by the appended claims and equivalents thereof. 

What is claimed is:
 1. A method for detecting CpG methylation of GPM6A (glycoprotein M6A) gene, the method comprising the steps of: (a) isolating genomic DNA from a clinical sample; (b) treating the genomic DNA or a fragment thereof with bisulfite; (c) amplifying a methylated CpG of GPM6A gene in the bisulfite-treated genomic DNA or fragment thereof from step (b) by using primer(s) to amplify a methylated CpG of the bisulfite-treated GPM6A gene; and (d) determining whether the CpG of GPM6A was methylated based on whether the DNA was amplified in step (c).
 2. The method of claim 1, wherein the step (c) is performed by detecting methylated CpG of the promoter or the CpG of the first intron of GPM6A gene.
 3. The method of claim 2, wherein the first intron of GPM6A gene has a sequence of SEQ ID NO:
 1. 4. The method of claim 1, wherein the detection of methylation is performed by a method selected from the group consisting of PCR, methylation-specific PCR, real-time methylation-specific PCR, PCR assay using a methylation DNA-specific binding protein, quantitative PCR, DNA chip-based assay, pyrosequencing, and bisulfite sequencing.
 5. The method of claim 1, wherein the clinical sample is selected from the group consisting of a tissue, cell, blood, blood plasma, serum, feces, and urine from a patient suspected of cancer or a subject to be diagnosed.
 6. The method of claim 1, wherein the primer(s) comprises at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of GPM6A.
 7. The method of claim 1, the primer(s) for amplifying a methylated CpG of GPM6A comprising sequence(s) having a homology of 50% or more with sequence(s) selected from the group consisting of SEQ ID NOs: 24-25, 27-106, 108-189, 191-392, 394-535, 537-658, 660-761, 763-844, 846-967, 969-1070, 1072-1193, 1195-1276, 1278-1359, and 1361-1408.
 8. The method of claim 1, further comprising probe(s) capable of hybridizing with a methylated CpG of GPM6A comprising at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of GPM6A.
 9. The method of claim 8, wherein the probe(s) comprising sequence(s) having a homology of 50% or more with sequence(s) selected from the group consisting of SEQ ID NOs: 26, 107, 190, 393, 536, 659, 762, 845, 968, 1071, 1194, 1277 and
 1360. 10. A kit for detecting CpG methylation of GPM6A (glycoprotein M6A) gene, comprising primer(s) to amplify a methylated CpG of the GPM6A gene.
 11. The kit of claim 10, wherein the primer(s) comprises at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of GPM6A.
 12. The kit of claim 10, wherein the primer(s) of step (c) comprises sequence(s) having a homology of 50% or more with sequence(s) selected from the group consisting of SEQ ID NOs: 24-25, 27-106, 108-189, 191-392, 394-535, 537-658, 660-761, 763-844, 846-967, 969-1070, 1072-1193, 1195-1276, 1278-1359, and 1361-1408.
 13. The kit of claim 10, further comprising probe(s) capable of hybridizing with a methylated CpG of GPM6A comprising at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of GPM6A.
 14. The kit of claim 10, wherein the probe(s) comprising sequence(s) having a homology of 50% or more with sequence(s) selected from the group consisting of SEQ ID NOs: 26, 107, 190, 393, 536, 659, 762, 845, 968, 1071, 1194, 1277 and
 1360. 